Compositions and methods for cytomegalovirus treatment

ABSTRACT

A panel of nucleic acids encoding human cytomegalovirus (HCMV) antigens is disclosed. These include nucleic acids encoding any one or more of the following antigenic proteins: glycoprotein B (gB), glycoprotein M (gM), glycoprotein N (gN), glycoprotein complex II (gM/N), glycoprotein complex III, phosphoprotein pp65 (pp65), phosphoprotein pp150 (pp150), and antigenic fragments of any of these. These nucleic acids can be used in kits and methods of treating HCMV infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Ser. No.60/450,818, filed Feb. 27, 2003, the contents of which are herebyincorporated by reference in its entirety.

TECHNICAL FIELD

[0002] This invention relates to compositions and methods of inhibitingand treating cytomegalovirus (CMV) infection using nucleic acidcompositions.

BACKGROUND

[0003] Human cytomegalovirus (HCMV) is a member of the family of herpesviruses. HCMV is endemic within the human population and infectionrarely causes symptomatic disease in immunocompetent individuals.However, HCMV infection of immunocompromised patients, including AIDSpatients and organ transplant recipients, can have serious consequences.HCMV infection has been shown to cause a variety of disorders in theimmunocompromised, including pneumonitis, retinitis, disseminatedviremia, and organ dysfunction. HCMV also poses a serious threat to thehealth of HIV-positive individuals because HCMV may accelerate thedevelopment of AIDS as well as contribute to the morbidity associatedwith increased immunodeficiency. Likewise, HCMV infection can beproblematic for pregnant women and children, especially infants andnewborns (Castillo and Kowalik, Gene, 290:19-34 (2002), Britt andAlford, Fields Virology, Third Ed. Chapter 77, pp. 2493-2523 (1996)).HCMV infection of newborns and the immunocompromised can be fatal.

[0004] Live attenuated vaccines have been used in the treatment of andprotection against HCMV. However, due to their ineffectiveness, a newstrategy is needed in designing a vaccine for HCMV.

SUMMARY

[0005] The invention features nucleic acid (e.g., naked DNA plasmid) CMV(e.g., HCMV) compositions that can elicit a broad immune response in therecipient. Specifically, neutralizing antibody responses, or both aneutralizing antibody immune response and a cell-mediated immuneresponse can be induced by immunization with one of more of the CMVconstructs of this panel. These compositions, or constructs, includenucleic acids (e.g., naked DNA plasmids) encoding antigenic proteins ofCMV (e.g., HCMV). Specifically, these antigenic proteins can includeglycoprotein B (gB)(also referred to as glycoprotein complex I (gcI)),glycoprotein M (gM), glycoprotein N (gN), glycoprotein complex II (gcIIor gM/N or gM/gN), glycoprotein complex III (gcIII or gH/L/O),phosphoprotein 65 (pp65), phosphoprotein 150 (pp150), and/or antigenicfragments (e.g., peptides) of any of these proteins. Once administeredto a recipient, the compositions cause cells in a recipient to produceantigenic proteins and peptides, which elicit a neutralizing antibodyimmune response, or both a neutralizing antibody immune response as wellas a cell-mediated immune response in the recipient.

[0006] In one aspect, the invention features compositions that include aplurality of sets of nucleic acid molecules, each set of nucleic acidmolecules encoding a different type of CMV polypeptide, and eachmolecule of a set encoding the same type of CMV polypeptide, wherein oneor more sets of the plurality encodes a CMV polypeptide that induces aneutralizing antibody response, and one or more sets of the pluralityencodes a CMV polypeptide that induces a cell-mediated immune response.

[0007] In various embodiments, the nucleic acid molecules are nucleicacid vectors (e.g., plasmids) which include elements that promoteexpression of the CMV polypeptide, such as a promoter that is operablylinked to the sequence encoding the CMV polypeptide (e.g., a CMVimmediate early (IE) promoter, or another promoter that drives CMV geneexpression in human cells), optionally, a leader peptide (e.g., a leaderpeptide which is expressed in frame with the CMV polypeptide, and whichcan replace a leader peptide encoded by the CMV gene), and apolyadenylation sequence (e.g., a bovine growth hormone polyA sequence).

[0008] In various embodiments, the CMV polypeptides encoded by thenucleic acids are human CMV polypeptides. The CMV polypeptides thatinduce an antibody response can be selected from the group ofglycoprotein B (e.g., gB of HCMV), glycoprotein complex II (e.g., gN andgM of HCMV), glycoprotein complex III (e.g., gH, gL, and gO of HCMV),and antigenic fragments thereof. In certain embodiments, the CMVpolypeptides that induce an antibody response are selected fromglycoprotein B and glycoprotein complex II, or antigenic fragmentsthereof.

[0009] The CMV polypeptides that induce a cell-mediated immune responsecan be selected from the group of phosphoprotein pp65 (pp65) (e.g., HCMVpp65), phosphoprotein pp150 (pp150) (e.g., HCMV pp150), and antigenicfragments thereof.

[0010] In another aspect, the invention features compositions thatincludes nucleic acid vectors encoding a plurality of types of HCMVpolypeptides that induce a neutralizing antibody response and optionallyone or more nucleic acid vectors encoding one or more types of HCMVpolypeptides that induce a cell-mediated immune response.

[0011] The invention also features compositions that include a pluralityof sets of nucleic acid vectors encoding HCMV polypeptides, each set ofnucleic acid vectors encoding a different type of HCMV polypeptide thatinduces a cell-mediated immune response. The compositions can furtherinclude a plurality of sets of nucleic acid vectors encoding HCMVpolypeptides, each set of nucleic acid vectors encoding a different typeof HCMV polypeptide that induces a neutralizing antibody response. Thepolypeptides that induce a neutralizing antibody response can beselected from the group of glycoprotein B, gM, gN, a combination of gMand gN (glycoprotein complex II; gcII), and a combination of gH, gL, andgO (glycoprotein complex III; gcIII) of HCMV, and antigenic fragmentsthereof. For example, the polypeptides that induce a cell-mediatedimmunity-inducing can include phosphoprotein 65, phosphoprotein 150,both phosphoprotein 65 and phosphoprotein 150, and/or antigenicfragments thereof.

[0012] The polypeptides that induce a neutralizing antibody response caninclude one of the following combinations: gcII or antigenic fragmentsthereof; gcIII or antigenic fragments thereof; gB and gcII or antigenicfragments thereof; gB and gcIII or antigenic fragments thereof.

[0013] In some aspects, the invention provides nucleic acid moleculesthat encode more than one CMV polypeptide (e.g., two, three, four, five,or six specific CMV polypeptides). Thus, the nucleic acid molecules of asingle set can encode one or more CMV polypeptides that induce aneutralizing antibody response (e.g., gcII or antigenic fragmentsthereof, gcIII or antigenic fragments thereof) and/or one or morepolypeptides that stimulate a cell-mediated immune response (e.g., pp65,pp150, or antigenic portions thereof).

[0014] In another aspect, the invention features compositions thatinclude a plurality of sets of nucleic acid molecules, each set ofnucleic acid molecules encoding a different type of CMV polypeptide thatinduces a neutralizing antibody response. In various examples, the CMVpolypeptides include gcII or antigenic fragments thereof; gcIII orantigenic fragments thereof; gB and gcII or antigenic fragments thereof;and gB and gcIII of HCMV or antigenic fragments thereof.

[0015] The invention also features pharmaceutical compositions thatelicit an immune response against HCMV. The compositions can include,for example, one or more of the nucleic acid compositions describedherein and a pharmaceutically acceptable carrier. The nucleic acidcompositions can include other features described herein.

[0016] The invention also features methods of and kits for eliciting animmune response against CMV (e.g., HCMV) in a subject. The methodsinclude, for example, administering to the subject an amount of apharmaceutical composition (e.g., a pharmaceutical composition describedherein) effective to elicit an immune response against CMV in thesubject. The immune response can be a protective immune response. Theadministration can be, e.g., by needle injection, needle-less jetinjection, gene gun, topical administration, surgical administration, ormucosal administration. The subject can be a non-human mammal or a human(e.g., a human sero-negative for HCMV, e.g., a female between the agesof 11 and 40, a female contemplating pregnancy, a pregnant female, anHIV-infected individual, a future organ transplant recipient, and afuture bone marrow donor, or, e.g., a human who is sero-positive forHCMV).

[0017] The new nucleic acid compositions (also referred to herein as DNAvaccines) have the advantages of having a combination of nucleic acidmolecules that can elicit a strong immune response against HCMV. Theyencode polypeptides that elicit a neutralizing antibody immune responseand/or nucleic acid molecules that encode polypeptides that elicit acell-mediated immune response against CMV. Certain combinations of thenucleic acid compositions contain a combination of nucleic acids thatwhen they express their encoded antigen or antigenic fragment can inducean unexpectedly strong neutralizing antibody response, e.g., in thecombination of gM/N and the combination of gM/N with gB. Thecombinations used in the new compositions elicit greater immuneresponses as a combination than as individual components alone.

[0018] Thus, these nucleic acid compositions have the advantage ofproviding broad immune protection against CMV (e.g., HCMV) usingdifferent immune mechanisms. The combination of eliciting neutralizingantibody immune response and cell-mediated immune response confersadvantages as a novel therapeutic agent for CMV (e.g., HCMV).Furthermore, combinations of certain antigens or antigenic fragmentsthat unexpectedly provide a strong immune response when combined alsoconfer advantages as a novel therapeutic agent for CMV (e.g., HCMV).

[0019] Exemplary amino acid and nucleic acid sequences for HCMV gB, gM,gN, gL, gH, gO, pp65, and pp150 are provided herein. In variousembodiments, a nucleic acid composition includes a gB nucleic acidsequence according to SEQ ID NO:1, or a sequence that is at least 75%,80%, 85%, 90%, 95%, 97%, 99%, or more identical to SEQ ID NO:1. Anucleic acid composition can encode a gB amino acid sequence that is75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical to SEQ ID NO:2. Thenucleic acid compositions can include a gM nucleic acid sequence that isat least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical to SEQ IDNO:3. The nucleic acid compositions can encode a gB amino acid sequencethat is at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical toSEQ ID NO:4.

[0020] A nucleic acid composition can include a sequence that is atleast 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical to a gNnucleic acid of SEQ ID NO:5, and/or the compositions can encode an aminoacid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, ormore identical to SEQ ID NO:6.

[0021] The nucleic acid compositions can include a sequence that is atleast 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical to a gHnucleic acid sequence according to SEQ ID NO:7, or the nucleic acids canencode an amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 97%,99%, or more identical to SEQ ID NO:8.

[0022] The nucleic acid compositions can include a sequence that is atleast 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical to a gLnucleic acid sequence according to SEQ ID NO:9, and/or the compositionscan encode an amino acid sequences at least 75%, 80%, 85%, 90%, 95%,97%, 99%, or more identical to SEQ ID NO:10.

[0023] The nucleic acid compositions can include a sequence that is atleast 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical to a gOnucleic acid according to SEQ ID NO:11, and/or the nucleic acids canencode an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%,99%, or more identical to SEQ ID NO:12.

[0024] The nucleic acid compositions can include a sequence that is atleast 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical to a pp65nucleic acid of SEQ ID NO:13, and/or the sequences can encode an aminoacid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or moreidentical to SEQ ID NO:14.

[0025] The nucleic acid compositions can include a sequence that is atleast 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more identical to a pp150nucleic acid of SEQ ID NO:15, and/or the compositions can encode anamino acid sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 99%, or moreidentical to SEQ ID NO:16.

[0026] The terms “gM/gN” or “gcII” refer to a nucleic acid compositioncontaining a combination of nucleic acids encoding gM and gN. Thus“gM/gN” and “gcII” are used interchangeably herein.

[0027] The terms “gcIII” or “gH/L/O” refer to a nucleic acid compositioncontaining a combination of nucleic acids encoding gH, gL, and gO, andare used interchangeably herein.

[0028] As used herein, “polypeptides” include both peptides andproteins. The term “CMV polypeptide” includes CMV polypeptides that arefull-length polypeptides encoded by a CMV gene, or antigenic portions orfragments thereof.

[0029] As used herein, the term “substantially identical” (or“substantially homologous”) refers to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleotide sequence such that the first and second amino acid ornucleotide sequences have at least 80% sequence identity. In the case ofantibodies, the second antibody has the same specificity and has atleast 50% of the affinity of the first antibody.

[0030] Calculations of “identity” between two sequences are performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 50% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which needs to be introducedfor optimal alignment of the two sequences.

[0031] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. The percent identity between two sequences is determinedusing the Needleman and Wunsch, J. Mol. Biol., 48:444-453, 1970,algorithm which has been incorporated into the GAP program in the GCGsoftware package, using a Blossum 62 scoring matrix with a gap penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0032] As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which isincorporated herein by reference. Aqueous and nonaqueous methods aredescribed in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: 1) lowstringency hybridization conditions: 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); 2) medium stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridizationconditions: 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one ormore washes at 0.2×SSC, 1% SDS at 65° C.

[0033] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0034] Other features, objects, and advantages of the invention will beapparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 is a schematic depiction of the different antigenicproteins encoded by nucleic acid constructs that can be used in avaccine against human cytomegalovirus (HCMV). Each bar represents thelength of the encoded amino acid sequence. DNA fragments of thesedifferent genes were subcloned into an optimized mammalian expressionvector pJW4303, which contains the CMV promoter. “gB” is the wild-typeglycoprotein B protein. “gBs” is the wild-type glycoprotein B proteintruncated at its carboxy terminus from 907 amino acids to 692 aminoacids, thus removing its transmembrane domain. “tPA.gBs” is thecarboxyterminal truncation of glycoprotein B containing the tPA leadersequence. “gM” is the wild-type glycoprotein M protein which is 372amino acids in length. “gN” is the wild-type glycoprotein N proteinwhich is 138 amino acids in length. “pp65” is the wild-typephosphoprotein protein which is 561 amino acids in length. “pp150” isthe wild-type phosphoprotein protein which is 1048 amino acids inlength. These antigenic proteins can be present in a number of differentcombinations or individually for inducing immune responses.

[0036]FIG. 2 is a graph showing that antisera raised by vaccination ofrabbits with gB and gBs have similar efficacy in neutralizing HCMV AD169virus (100 pfu). Both constructs induced neutralizing antibody responseto AD169 viral infection as seen by the percent reduction of infection.The x-axis is antisera dilutions from 1 to 1024 and the y-axis is thepercent reduction of HCMV infection as measured by reduction of theinfected CV-1 cell nuclei.

[0037]FIGS. 3A and 3B are western blots showing recognition of gM (FIG.3A) or gN (FIG. 3B) in 293T cells transfected with the following DNAvaccines: gM+gN (lane3), gM (lane 4) or gN (lane 5). CMV virion samplesgrown from FSK cells (lane 1) were used as positive controls anduninfected FSK cells (lane 2) or 293T cells transfected with empty DNAvaccine vector (lane 6) were included as the negative controls. Mousemonoclonal antibodies against gM (FIG. 3A) or gN (FIG. 3B) were used totest the expression of these DNA vaccines.

[0038]FIG. 4 is a western blot showing the specificity of anti-gM/gNantibody responses in sera from rabbits immunized with the gM+gN DNAvaccine. Lane 1 contains lysate of HCMV infected FSK cells (positivecontrol). Lane 2 contains lysate of non-infected FSK cells (negativecontrol). Lane 3 contains lysate from 293T cells transfected with gM+gN.Lane 4 contains lysate from 293T cells transfected with gM. Lane 5contains lysate from 293T cells transfected with gN. Lane 6 containslysate from cells tranfected with empty vector (negative control).

[0039]FIG. 5 is a western blot showing specific antigen (lane a)recognition by a 1:100 fold dilution of serum from immunized Balb/Cmice. The clean background seen in the control lane (lane b) suggeststhat the immune response to pp65 is highly specific.

[0040] FIGS. 6A-D are a series of plots of a FACS result ofintracellular staining (ICS) analysis of splenocytes from mice immunizedwith pp65 naked DNA plasmid vaccine. The first three plots, FIGS. 6A-C,are results of a FACS analysis of splenocytes from a mouse immunizedwith a pp65 vaccine. The fourth plot, FIG. 4D, is the negative controlshowing the results of a FACS analysis of splenocytes from a mouseimmunized with vector control alone. CD8 expression is plotted on thex-axis. IFN-γ expression is plotted on the y-axis.

[0041] FIGS. 7A-D are a series of plots of a FACS result of ICS analysisof splenocytes from mice immunized with pp150 naked DNA plasmid vaccine.The first three plots, FIGS. 7A-C, are results of a FACS analysis ofsplenocytes from a mouse immunized with the pp150 DNA vaccine. Thefourth plot, FIG. 7D, is the negative control showing the results ofsplenocytes from a mouse immunized with vector control alone. CD8expression is plotted on the x-axis. IFN-γ expression is plotted on they-axis.

[0042]FIG. 8A is a schematic depiction of the different gcIII antigenicproteins encoded by nucleic acid constructs that can be used in avaccine against human cytomegalovirus (HCMV). Each bar represents thelength in amino acid sequence encoded. DNA fragments of these differentgenes were subcloned into an optimized mammalian expression vectorpJW4303, which contains the CMV promoter. “gH” is the wild-typeglycoprotein H protein which is 743 amino acids in length. “gL” is thewild-type glycoprotein L which is 278 amino acids in length. “gO” is thewild-type glycoprotein O protein which is 466 amino acids in length.These antigenic proteins can be present in a number of differentcombinations or individually for inducing immune responses.

[0043]FIG. 8B is a western blot showing the recognition of the gHprotein in various samples. Lane 1 contains lysate of HCMV infected FSKcells (positive control). Lane 2 contains lysate of non-infected FSKcells (negative control). Lane 3 contains lysate from 293T cellstransfected with gH. Lane 4 contains lysate from 293T cells transfectedwith gH+gL. Lane 5 contains lysate from 293T cells transfected withgH+gL+gO. Lane 6 contains lysate from 293T cells tranfected withgH+gL+gB. Lane 7 contains lysate from 293T cells transfected withgH+gL+gO+gB. Lane 8 contains lysate from 293T cells transfected withempty vector (negative control).

[0044]FIG. 8C is a western blot showing the recognition of the gLprotein in various samples. Lane 1 contains lysate of HCMV infected FSKcells (positive control). Lane 2 contains lysate of non-infected FSKcells (negative control). Lane 3 contains lysate from 293T cellstransfected with gH. Lane 4 contains lysate from 293T cells transfectedwith gH+gL. Lane 5 contains lysate from 293T cells transfected withgH+gL+gO. Lane 6 contains lysate from 293T cells tranfected withgH+gL+gB. Lane 7 contains lysate from 293T cells transfected withgH+gL+gO+gB. Lane 8 contains lysate from 293T cells transfected withempty vector (negative control).

[0045]FIGS. 9A and 9B are a set of western blots showing recognition ofthe gH (FIG. 9A, upper panel) and gL (FIG. 9B, lower panel) proteins invarious sample. “L” and “S” directly under each lane refer to samplesfrom cell lysates and supernatants, respectively. gH/L/O refers tosamples in which gH, gL, and gO were co-expressed. gH/gL refers tosamples from cells in which gH and gL were co-expressed. gH/O refers tosamples from cells in which gH and gO were co-expressed. gH refers to asample from cells in which gH was expressed. Vec refers to a sample inwhich empty vector was transfected.

[0046]FIG. 10 is a representation of a HCMV gB nucleic acid sequence(SEQ ID NO:1).

[0047]FIG. 11 is a representation of a HCMV gB amino acid sequence (SEQID NO:2).

[0048]FIG. 12 is a representation of HCMV gM nucleic acid and amino acidsequences (SEQ ID NO:3 and SEQ ID NO:4, respectively).

[0049]FIG. 13 is a representation of HCMV gN nucleic acid and amino acidsequences (SEQ ID NO:5 and SEQ ID NO:6, respectively).

[0050]FIG. 14 is a representation of a HCMV gH nucleic acid sequence(SEQ ID NO:7).

[0051]FIG. 15 is a representation of a HCMV gH amino acid sequence (SEQID NO:8).

[0052]FIG. 16 is a representation of HCMV gL nucleic acid and amino acidsequences (SEQ ID NO: 9 and SEQ ID NO:10, respectively).

[0053]FIG. 17 is a representation of HCMV gO nucleic acid and amino acidsequences (SEQ ID NO:11 and SEQ ID NO:12, respectively).

[0054]FIG. 18 is a representation of HCMV pp65 nucleic acid and aminoacid sequences (SEQ ID NO:13 and SEQ ID NO:14, respectively).

[0055]FIG. 19 is a representation of a HCMV pp150 nucleic acid sequence(SEQ ID NO:15).

[0056]FIG. 20 is a representation of a HCMV pp150 amino acid sequence(SEQ ID NO:16).

[0057] Like reference symbols in the various drawings indicate likeelements.

DETAILED DESCRIPTION

[0058] The invention provides a panel of nucleic acids that can be usedto inhibit or treat CMV (e.g., HCMV). This panel includes nucleic acidcompositions (e.g., naked DNA plasmid vectors) encoding antigenicproteins of HCMV (e.g., of strain AD169). Specifically, these antigenicproteins can include glycoprotein B (gB or also referred to asglycoprotein complex I (gcI)), glycoprotein M (gM), glycoprotein N (gN),a combination of gM and gN called glycoprotein complex II (gcII or gM/Nor gM/gN), a combination of gH, gL, and gO called glycoprotein complexIII (gcIII or gH/L/O), phosphoprotein pp65 (pp65), phosphoprotein pp150(pp150), or antigenic fragments (e.g., peptides) of any of these.Antigenic proteins of HCMV can include those that induce a neutralizingantibody immune response (e.g., gB, gM, gN, gcII, and gcIII). Antigenicproteins of HCMV also include those that induce a cell-mediated immuneresponse (e.g., pp65 and pp150). The new vaccines can include DNAencoding more than one of these antigenic proteins or fragments.

[0059] For example, the compositions can include nucleic acid molecules(e.g., naked DNA plasmids) encoding gcII (gM/gN) and/or gcIII (gH/L/O).In other examples, the compositions can include nucleic acid moleculesencoding gB in combination with either or both nucleic acid moleculesencoding gcII and nucleic acid molecules encoding gcIII. Alternatively,the compositions can include nucleic acid molecules encoding gB, gcII,and pp65. Another example is a composition including nucleic acidmolecules encoding gB, gcII, and pp150. In yet another example, acomposition can include nucleic acid molecules encoding gB, gcII, pp65,and pp150. Any number of combinations of DNA encoding polypeptidecapable of inducing neutralizing antibody with or without DNA plasmid(e.g., naked DNA plasmid) encoding polypeptide capable of inducingcell-mediated immune response can be used for the CMV (e.g., HCMV)vaccines of the invention.

[0060] The new nucleic acid compositions can induce a protective immuneresponse in an individual (i.e., protection against a subsequentexposure to HCMV that inhibits or prevents an HCMV infection). Thisimmune response can include humoral immunity or cell-mediated immunity,or both. Humoral immunity involves the induction of neutralizingantibodies against the infecting organism (e.g., CMV, e.g., HCMV).Cell-mediated immunity involves a cytotoxic T lymphocyte response to theinfecting organism (e.g., CMV, e.g., HCMV). Humoral immunity can beinduced by gB, gcII, gcIII, or any combination of these, antigenicfragments of these, or antigenic fragment combinations of these.Cell-mediated immunity can be elicited by pp65 and/or pp150 or antigenicfragments of these.

[0061] The new nucleic acid compositions combine antigens that elicitneutralizing antibody immune response to CMV (e.g., HCMV) and antigensthat elicit cell-mediated immune response to CMV (e.g., HCMV) in asingle composition. Administration of any one of these compositions caninclude a prime or boost with attenuated virus vaccine and/orpolypeptide vaccine, or may not include either of these supplements.

[0062] Human Cytomegalovirus Proteins

[0063] The invention is based, in part, on the discovery thatadministering nucleic acids encoding HCMV proteins in particularcombinations promotes expression of the proteins and can enhance immuneresponses to the virus. Expressing these combinations can stimulate bothhumoral and cell-mediated immune responses, thus providing potentbroad-spectrum immunity. Providing HCMV glycoprotein expression via DNAadministration has the added benefit of allowing biosynthesis andassembly of the proteins in human cells, and eliminates complications ofglycoprotein expression and purification ex vivo. Because theglycoproteins provided herein can be expressed within the cells of asubject, they can include membrane-associated domains, glycosylation,and other post-translational modifications which confer immunogenicproperties on the polypeptides.

[0064] Exemplary nucleic acid and amino acid sequences of HCMV gN, gM,gL, gH, gO, gB, pp65, and pp150 are provided herein (see, e.g., FIGS.10-20, and SEQ ID NOs:1-16). The compositions described herein caninclude and/or encode sequences that are at least 75%, 80%, 85%, 90%,95%, 97%, 99%, or more identical to one or more of SEQ ID NOs:1-16. Inaddition, or alternately, the compositions described herein can includesequences that are from a laboratory-adapted strain (e.g., AD169, Towne)or a natural isolate. Natural isolates can be derived from infectedpatients by methods known in the art (see, e.g., Walker et al., J. Clin.Microbio., 39:2219-2226,2001 and references cited therein for discussionof sequence variability and method of isolating and comparing HCMVstrains).

[0065] The nucleic acid compositions herein can encode full-length HCMVgene products, or may include antigenic fragments thereof. Antigenicfragments are fragments that are immunogenic when administered to ananimal (e.g., under conditions that promote an immune response, e.g.,with an adjuvant, or, e.g., when expressed from a DNA vaccine).Antigenic fragments are at least 10, 20, or 30 amino acids in length, orrepresent a specific domain (e.g., a soluble portion of a membraneglycoprotein). Methods for determining whether a fragment is immunogenicare known. In one example, a nucleic acid composition encoding afragment of a HCMV protein is administered to an animal, followed by 2-4boosts of the nucleic acid at periods of 2-4 weeks thereafter. Serumfrom the animal is isolated and tested for reactivity with the proteinencoded by the nucleic acid (e.g., reactivity with a transfected celllysate by western blot). Methods for testing cell-mediated immunity arealso known, and include assays to measure cytokine release and/orproliferation of lymphocytes in response to stimulation with antigen.

[0066] Nucleic Acid Compositions

[0067] Nucleic acid compositions are useful in inhibiting, e.g.,preventing, or controlling infection or disease by inducing immuneresponses, to an antigen or antigens, in an individual. For example,nucleic acid compositions can be used prophylactically in naiveindividuals, or therapeutically in individuals already infected with CMV(e.g., HCMV). Traditional vaccines, which include inactivated viruses orsubunit protein or peptide antigen, have had poor immunogenicity andpoor induction of cell-mediated immunity, and low efficacy. In addition,live attenuated vaccines can have safety concerns. A major advantage ofnucleic acid-based compositions is that they can induce both antibodyand cell-mediated immune responses. The development of nucleic acidvaccines has proved to be promising, and they can be administered incombination with or as a boost to traditional inactivated virus and/orsubunit protein or peptide antigen.

[0068] The nucleic acid compositions described herein have the advantageof containing a combination of nucleic acid molecules that encodepolypeptides that elicit neutralizing antibody immune response andnucleic acid molecules that encode polypeptides that elicitcell-mediated immune response against CMV (e.g., HCMV). Variouscombinations of nucleic acids described herein elicit greater immuneresponses in combination than as individual components alone. In certainembodiments, the combinations can provide a greater than additive (orsynergistic) effect. Thus, these compositions have the advantage ofproviding broad immune protection against CMV, (e.g., HCMV) usingdifferent immune mechanisms. This combination of eliciting neutralizingantibody immune response and cell-mediated immune response confersadvantages for inhibition of CMV (e.g., HCMV).

[0069] Nucleic acid compositions for inducing immune responses canconsist of naked DNA plasmids that express the antigen. Bacterialvectors, replicon vectors, live attenuated bacteria, DNA vaccineco-delivery with live attenuated vectors, and viral vectors forexpression of heterologous genes also can be used. Bacterial vectorssuch as BCG and Listeria have been used and show promise. In the case ofnaked DNA replicon vectors, a mammalian expression plasmid serves as avehicle for the initial transcription of the replicon.

[0070] The replicon is amplified within the cytoplasm, resulting in moreabundant mRNA encoding the heterologous gene such that initialtransfection efficiency may be less important for immunogenicity. Liveattenuated viral vectors (e.g., recombinant vaccinia (e.g., modifiedvaccinia Ankara (MVA), IDT Germany), recombinant adenovirus, avianpoxvirus (e.g., canarypox (e.g., ALVAC®, Aventis Pasteur) or fowlpox),poliovirus, and alphavirus virion vectors) have been successful ininducing cell-mediated immune response and can be used as well. Theavian poxviruses are defective in mammalian hosts, but can expressinserted heterologous genes under early promoters. Recombinantadenovirus and poliovirus vectors can thrive in the gut and so canstimulate efficient mucosal immune responses. Finally, attenuatedbacteria as a vehicle for DNA vaccination delivery has been useful.Examples include S. enterica, S. tymphimurium, Listeria, and BCG. Theuse of mutant bacteria with weak cell walls can aid the exit of DNAplasmid from the bacterium.

[0071] DNA uptake can sometimes be improved by the use of theappropriate adjuvants. Synthetic polymers (e.g., polyamino acids,co-polymers of amino acids, saponin, paraffin oil, muramyl dipeptide,Regressin (Vetrepharm, Athens Ga.), and Avridine) and liposomalformulations can be added as adjuvants to the vaccine formulation toimprove DNA stability and DNA uptake by the host cells, and may decreasethe dosage required to induce an effective immune response. Regardlessof route, adjuvants can be administered before, during, or afteradministration of the nucleic acid. Not only can the adjuvant increasethe uptake of nucleic acid into host cells, it can increase theexpression of the antigen from the nucleic acid within the cell, induceantigen presenting cells to infiltrate the region of tissue where theantigen is being expressed, or increase the antigen-specific responseprovided by lymphocytes.

[0072] Nucleic acid uptake can be improved in other ways as well. Forexample, DNA uptake via IM delivery of vaccine can be improved by theaddition of sodium phosphate to the formulation. Increased DNA uptakevia IM delivery can also be accomplished by electrotransfer (e.g.,applying a series of electrical impulses to muscle immediately after DNAimmunization). Adjuvants which can also be added to the vaccine toimprove DNA stability and uptake as well as improve immune inductioninclude water emulsions (e.g., complete and incomplete Freund'sadjuvant), oil, Corynebacterium parvum, Bacillus Calmette Guerin, ironoxide, sodium alginate, aluminum hydroxide, aluminum and calcium salts(i.e., alum), unmethylated CpG motifs, glucan, and dextran sulfate.Coinjection of cytokines, ubiquitin or costimulatory molecules can alsohelp improve immune induction. Fusions of the antigen with cytokinegenes, helper epitopes, ubiquitin, or signal sequences have beensuccessful and can also induce immune response. Fusions that aid intargeting to certain cell types can also be done. For example, antigenfused to L-selectin was successful in targeting the antigen to highendothelial venules of peripheral lymph nodes.

[0073] The medium in which the DNA vector is introduced should bephysiologically acceptable for safety reasons. Suitable pharmaceuticalcarriers include sterile water, saline, dextrose, glucose, or otherbuffered solutions (e.g., TE or PBS). Included in the medium can bephysiologically acceptable preservatives, stabilizers, diluents,emulsifying agents, pH buffering agents, viscosity enhancing agents,colors, etc.

[0074] Once the DNA vaccine is delivered, the nucleic acid molecules(e.g., DNA plasmids) are taken up into host cells, which then expressthe plasmid DNA as protein. Once expressed, the protein is processed andpresented in the context of self-major histocompatibility complex (MHC)class I and class II molecules. An immune response is then generatedagainst the DNA-encoded immunogen. To improve the effectiveness of thevaccine, multiple injections can be used for therapy or prophylaxis overextended periods of time. To improve immune induction, a prime-booststrategy can be employed. Priming vaccination with DNA and a differentmodality for boosting (e.g., live viral vector or protein antigen) hasbeen used successfully in inducing cell-mediated immunity. The timingbetween priming and boosting varies and is adjusted for each vaccine.

[0075] Administering Nucleic Acid Compositions

[0076] The new nucleic acid compositions can be administered, orinoculated, to an individual as naked nucleic acid molecules (e.g.,naked DNA plasmids) in physiologically compatible solutions such aswater, saline, Tris-EDTA (TE) buffer, or in phosphate buffered saline(PBS). They can also be administered in the presence of substances(e.g., facilitating agents and adjuvants) that have the capability ofpromoting nucleic acid uptake or recruiting immune system cells to thesite of inoculation. Nucleic acid compositions for inducing immuneresponses have many modes and routes of administration. They can beadministered intradermally (ID), intramuscularly (IM), and by eitherroute, they can be administered by needle injection, gene gun, orneedle-less jet injection (e.g., Biojector™ (Bioject Inc., Portland,Oreg.). Other modes of administration include oral, intravenous,intraperitoneal, intrapulmonary, intravitreal, and subcutaneousinoculation. Modes of mucosal vaccination may also be employed. Theseinclude delivery, for example, via intranasal, ocular, oral, vaginal, orrectal topical routes. Delivery by these topical routes can be by nosedrops, eye drops, inhalants, suppositories, or microspheres. Alsopossible are delivery methods which use an electroporation device, orwhich rely on skin surface absorption.

[0077] Suitable doses of nucleic acid compositions for humans can rangefrom 1 μg/kg to 1 mg/kg of total nucleic acid in a composition, e.g.,from 5 μg/kg-500 mg/kg of a nucleic acid composition, 10 μg/kg-250 μg/kgof a nucleic acid composition, or 10 μg/kg-170 μg/kg of a nucleic acidcomposition. In one embodiment, a human subject (18-50 years of age,45-75 kg) is administered 1.2 mg-7.2 mg of a nucleic acid composition.The nucleic acid composition includes compositions containing a pool ofnucleic acids encoding distinct antigens. For example, a dose of 3 mg ofa nucleic acid composition encoding 3 different CMV antigens can have 1mg of DNA encoding each antigen. DNA vaccines can be administeredmultiple times, e.g., between two-six times, e.g., three times. In anexemplary method, 100 μg of a DNA composition is administered to a humansubject at 0, 4, and 12 weeks (100 μg per administration).

[0078] Assessing Immune Responses Induced by Nucleic Acid Compositions

[0079] Advancements in the field of immunology have allowed morethorough and sensitive evaluation of cellular responses to candidateCMV, e.g., HCMV, vaccines. Such assays as Intracellular CytokineStaining (ICS) and ELISPOT (an enzyme-linked immunosorbent assayformat), allow detecting and counting cells producing cytokines (e.g.,TNFα and IFN-γ) in response to antigen. For example, isolation ofsplenocytes or peripheral blood monocyte cells (PBMCs) from animals orhuman patients followed by in vitro stimulation with HCMV epitopes suchas recombinant vaccinia virus expressing pp65 or pp150, and finallytesting by ELISPOT and/or intracellular cytokine staining (ICS) candetermine the potential for inducing a cell-mediated immune response ina vaccine recipient. Flow cytometry using tetramers (i.e., moleculesconsisting of four copies of a given class I molecule bound to theircognate peptide and alkaline phosphatase) allows the enumeration ofantigen-specific T cells (e.g., detection of T cells that recognizespecific peptides bound to major histocompatibility complex (MHC) classI molecules). A chromium release assay allows the assessment ofcytotoxicity. To assess a cell-mediated immune response to a DNAvaccine, the more traditional approaches of measuring T cellproliferation in response to antigen and CTL-mediated killing ofautologous cells expressing CMV, e.g., HCMV epitopes are also available.

[0080] ELISA assays and Western blots have allowed the assessment ofhumoral immune responses. ELISA and Western blots can be used to assessantibody binding and antibody specificity. Neutralizing assays candetermine the ability of antibody to protect against HCMV infection.

[0081] Neutralizing Antibody Responses Induced by gM and gM/gN DNAVaccines

[0082] While the gM DNA vaccine was able to induce a neutralizingantibody response from a serum dilution of 1:64 and gN was unable toinduce a detectable response due to its low expression rate, inimmunized rabbits, the combination vaccine of gM and gN DNA was able toinduce a much higher neutralizing antibody response (a serum dilution of1:256) than expected. gM in combination with gN unexpectedly induced agreater than additive (synergistic) neutralizing antibody response (SeeTable 1, Example 2).

[0083] The combination of antisera raised by gB plus gM/gN furtherimproved the neutralizing activity over using either antisera raised bygB alone or antisera raised by gM/N alone. (See Table 1, Example 2.)

[0084] This new information will have significant impact in thedevelopment of human CMV vaccines.

[0085] Kits

[0086] The invention also includes kits comprising the nucleic acidcompositions described herein. The kits can include one or more otherelements including: instructions for use; other reagents, e.g., adiluent, devices or other materials for preparing the composition foradministration; pharmaceutically acceptable carriers; and devices orother materials for administration to a subject. Instructions for usecan include instructions for therapeutic application (e.g., DNAvaccination and boosting) including suggested dosages and/or modes ofadministration, e.g., in a human subject, as described herein.Instructions can also provide directions for prophylactic treatment,e.g., in patients who are susceptible to HCMV infection, e.g., asdescribed herein.

[0087] The kit can further contain at least one additional reagent, suchas a diagnostic or therapeutic agent, e.g., a diagnostic agent tomonitor a response to immune response to the compositions in thesubject, or an additional therapeutic agent.

[0088] In one embodiment, the kit includes a vial (or other suitablecontainer) containing nucleic acids encoding two, three, four, five, orsix distinct CMV antigens (e.g., gcII antigens, gcIII antigens, gB,pp65, pp150, or antigenic fragments thereof). In various examples, thekit includes nucleic acids encoding one of the following combinations ofCMV antigens: gcII and pp65; gcIII and pp65; gcII and pp150; gcIII andpp150; gcII, pp65, and pp150; gB, gcII, and pp65, and so on.

[0089] Administration of Vaccines to Humans

[0090] A person sera-negative for HCMV can be immunized with one or acombination of vaccines described herein. One primary objective of a CMVvaccine is to inhibit infection during pregnancy. This would implyvaccination of all sera-negative adolescent girls or older women whointend to become pregnant (e.g., greater than age 11, e.g., between theages of 11 to 40). These include a female contemplating pregnancy or apregnant female. Vaccination can be advised just before pregnancy iscontemplated. Regular boosts can be given throughout childbearing years.Both adolescent and pediatric vaccination can be considered. Anotherobjective of an HCMV vaccine is to induce or enhance immunity in organ(e.g., kidney, liver, heart, and lung) transplant recipients beforetransplant surgery or to induce or enhance immunity in HIV-infectedindividuals. Another objective is to induce immunity in bone marrowdonors so they can serve as sources of transfused T cells active againstHCMV. Another objective is to administer the vaccine to sero-positiveindividuals as a therapeutic application against current HCMV infection.

[0091] Formulations

[0092] Two or more immunizations may be beneficial. HCMV DNA vaccinescan be delivered individually or in combination with other HCMV DNAvaccines. The multi-antigen formulation can be delivered as a premixedformulation or in multiple separate inoculations, that are administeredclose enough together in time that all antigens are expressed andpresent in the recipient at the same time.

[0093] The new HCMV DNA vaccines can be used alone or in combinationwith additional modalities of HCMV vaccines, e.g., live-attenuated,killed, protein or peptide based, viral or bacterial vector based can bedelivered. Such a combination can be delivered sequentially(prime+boost) or concurrently in the same or more than one inoculationsimultaneously. The new HCMV DNA vaccines can be used as either thepriming or boosting component or both.

[0094] The HCMV gene inserts in the HCMV DNA vaccines of the inventioncan also be used for other forms of HCMV vaccine productions. Forexample, the HCMV gene inserts can be cloned into other viral orbacterial vectors. The new HCMV gene inserts can also be cloned into arecombinant protein expression system to produce HCMV proteins forvaccine and diagnostic reagents. Suitable plasmids, deliver, and dosagesof nucleic acid compositions are described elsewhere herein. In variousexamples, suitable plasmids for administration in humans will containpromoters which permit high levels of expression of the CMV antigens(e.g., promoters that are highly active and/or constitutively active inhuman cells). HCMV promoters (e.g., the HCMV immediate early promoter)can be used to drive expression of the HCMV antigens.

EXAMPLES

[0095] The invention is further described in the following examples,which do not limit the scope of the invention described in the claims.

[0096] The nucleic acid compositions described herein have the advantageof being able to raise broad immune responses, including neutralizingantibody responses and cellular immune responses against multiple CMV(e.g., HCMV) antigens. For the following examples, cDNA fragments ofdifferent CMV (e.g., HCMV) genes (e.g., gB, truncated gB (gBs),truncated gB containing a tPA leader sequence (tPA.gBs), gM, gN, gcI,gcII, gcIII, pp65, and pp150) were subcloned into an optimized mammalianexpression vector, pJW4303, which uses the CMV promoter (Lu et al.,Methods in Molecular Medicine, 29:355-374). Briefly, the pJW4303 vectorcontains, in the following order, CMV immediate early (IE) promoter (bp1-1194), CMV IE promoter intron A (bp 1195-2027), tissue plasminogenactivator (tPA) (bp2027-2102), bovine growth hormone (BGH) polyA (bp2119-2419), pBR vector backbone (amp R) (2419-4794), and SV40 Ori (bp4794-5139). cDNA fragments of the genes to be expressed can be insertedinto the site formed by digestion with HindIII (2027) and BamHI (2119)or the site formed by digestion with NheI (2102) and BamHI (2119).

[0097]FIG. 1 shows some of the CMV genes gB, gBs, tPA.gBs, gM, gN, pp65,and pp150, which were cloned into the pJW4303 optimized vector andtested in animals (e.g., rabbits and Balb/C mice). These naked DNAplasmids were used as vaccines either individually or in combination.

Example 1 gB DNA Vaccine

[0098] Rabbits were immunized by gene gun with the gB DNA vaccine (6 μg)or vector control vaccine (6 μg) three times at intervals of four weekseach. Two weeks after the third immunization, blood was drawn from therabbit and serum was isolated from the blood. Serum was then tested forability to neutralize AD169.

[0099] Neutralization Assay

[0100] Flat bottomed 96-well microtiter plates were seeded with 2-5×10⁴FSK cells 2-3 days prior to performing the neutralization assay. Morethan 80% confluence is ideal for conducting the neutralizing assay. Atday 1, viral stock was incubated at 37° C. for 1 hour 0.3 ml ofinfectious virus (approximately 150 PFU) was mixed with 0.3 ml ofdiluted natural rabbit antisera (both postbleed and prebleed or negativesera). Also medium without virus was included as positive control, and1000 pfu/well was included for positive control. After 60 minutes ofincubation at 37° C., 0.2 ml of a virus-antibody mixture was added toreplicate wells of a 96-well tissue culture plate containing FSK cells.Three hours later, the inoculum was removed and the monolayer was fedwith fresh complete medium and incubated 16 hours.

[0101] The following day, the medium was removed and the monolayer waswashed once with Dulbecco's phosphate-buffered saline, pH 7.4, and thenfixed in absolute ethanol for 20 minutes at room temperature. Followingfixation, the monolayer was immediately rehydrated with DPBS. The cellswere than stained with 50 μl of tissue culture medium containing MAbp63-27, which is reactive against immediate-early gene 1 product pp72,for 40 minutes at 37° C. After washing the wells twice with DPBS, 50 μlof fluorescein conjugated goat anti-mouse IgG diluted 1/60 in DPBS wasadded and the plate incubated for 40 minutes. The wells were then washedtwice with DPBS, counterstained with 0.02% Evans blue and 0.2 ml of DPBScontaining 30% glycerol was added. Finally, the plate was viewed withLeitz epifluorescence microscope with a stage adapter for readingmicrotiter plate. Fluorescent nuclei were counted. Infectivity wasexpressed as fluorescent nuclei per ml of inoculum. Neutralization wasexpressed as the mean percent reduction in fluorescent nuclei comparedwith control cultures containing no antibody or control nonimmune sera.50% reduction was used as the indicator.

[0102]FIG. 2 is a graph showing neutralization of HCMV AD169 viruseswith antisera dilutions. As seen in FIG. 2, both gB and gBs constructsinduced neutralizing antibody responses, with almost 100% reduction ofinfection seen with antisera dilution of 1:16. Previously, gBs (whichlacks the transmembrane domain) was more commonly used due to solubilityissues with the full length gB (which retains the transmembrane domain).Full-length gB in the optimized pJW4303 construct was successful here asseen in FIG. 2.

Example 2 Co-Expression Enhances Production of gM and gN

[0103] 293T cells were transiently tranfected with a gM and gN-encodingDNA vaccine plasmid, a gM-encoding DNA vaccine plasmid, or a gN-encodingDNA vaccine plasmid. Lysates of transfected cells were analyzed forexpression of gM and gN by western blotting. The results are depicted inFIGS. 3A and 3B. Lysates of cells transfected with gM alone (10 μg ofDNA was transfected; FIG. 3A, lane 4, and FIG. 3B, lane 4) and gN alone(10 μg of DNA was transfected; FIG. 3A, lane 5, and FIG. 3B, lane 5) didnot exhibit expression of gM or gN by western blotting. However, lysatesof cells transfected with gM- and gN-encoding DNA did exhibit expressionof each by western blotting (10 μg of each DNA was transfected; FIG. 3A,lane 3, and FIG. 3B, lane 3). Lysates of CMV-infected cells (positivecontrol (FIG. 3A, lane 1, and FIG. 3B, lane 1) displayed expression ofboth gM and gN. This experiment shows that co-expression of DNA encodingboth gM and gN resulted in higher levels of expression than observedwhen either glycoprotein was expressed alone. gM and gN, whenco-expressed, form a disulfide-linked complex. Complex formation may berequired for transport of the proteins from the endoplasmic reticulum toGolgi and trans-Golgi compartments (Mach et al., J. Virol.,74(24):11881-11892, 2000). Co-administration of gM- and gN-expressingnucleic acids may permit optimal expression of these gene products,leading to potent stimulation of an immune response against theantigens. Furthermore, providing these antigens via DNA vaccinationeliminates the complications of preparing proteins for administration.This is particularly useful for proteins such as these, which aremembrane-associated and form complexes during biosynthesis in the cell,and thus would be expensive to express, purify, and administer inprotein form.

Example 3 gM, gN, gcII and gB+gcII DNA Vaccines

[0104] Rabbits were immunized by gene gun with the gB DNA vaccineplasmid (6 μg), the gM vaccine plasmid, the gcII (gM/N) vaccine plasmid,gB+gcII vaccine plasmid, or vector control vaccine plasmid three timesat intervals of four weeks each. Two weeks after the third immunization,blood was drawn from the rabbit and serum was isolated from the blood.

[0105] Sera were tested for recognition of gM and gN by westernblotting. As shown in FIG. 4, sera from rabbits immunized with the gcIIplasmid recognized protein expressed in HCMV-infected FSK cells (FIG. 4,lane 1), and gM and gN expressed in 293T cells (FIG. 4, lane 3). Asnoted above, the gN plasmid did not give rise to a detectable responsein other tests.

[0106] Sera were also tested for neutralization of AD169. The protocolfor the neutralization assay is described in Example 1, above. As seenin Table 1, while the gM vaccine alone was able to induce low titerneutralizing antibody in the immunized rabbit (and gN alone had beenshown to be ineffective), the combination of gM and gN (gM/N) was ableto induce a much higher, and greater than additive (synergistic),neutralizing antibody response. A higher neutralizing titer is reflectedin the fact that virus was neutralized by a greater dilution of serumfrom gM/N-vaccinated rabbits as compared to rabbits vaccinated with gMalone. Thus, these two antigenic proteins can produce a neutralizingantibody response in the rabbit. Furthermore, the combination of gB andgM/N was able to induce a protective antibody response. These dataindicate that vaccination with a combination of gB and gM/N can producea neutralizing antibody response in rabbits that is at least additiveand can therefore provide a protective response. TABLE 1 Neutralizationof AD169 (50 pfu) with DNA vaccine induced rabbit sera rabbit sera 50%reduction (dilution) Anti-gB 1:256 Anti-gM 1:64  Anti-gM/N 1:256Anti-gB + Anti-gM/N 1:512 vector control 1:8 

Example 4 pp65 DNA Vaccine

[0107] Balb/C mice were immunized by gene gun three times at four-weekintervals with DNA vaccine expressing human CMV pp65. 6 μg of DNA wasadministered at each immunization. Two weeks after the thirdimmunization, blood was drawn and serum was isolated from the blood andtested. As seen in FIG. 5, a 1:100 dilution of serum from mice immunizedwith the pp65 vaccine was able to specifically recognize antigen bywestern blot analysis.

[0108] At a time within 2 weeks and 6 months after the thirdimmunization of the mice, cell-mediated immunity was tested. On the dayof actual testing, the immunized mice were sacrificed, the spleen wasremoved and splenocytes were isolated after red blood cell lysis.Splenocytes were then tested by specific stimulation with related HCMVantigen, namely recombinant vaccinia virus expressing pp65 (VV/pp65).

[0109] FIGS. 6A-D show the results of experiments in which mice wereimmunized with the pp65 vaccine. Immunized mice were capable of mountinga specific cell-mediated response to vaccinia virus VV/p65 stimulationin culture. The first three plots (FIGS. 6A-C) are results of a FACSanalysis of splenocytes from a mouse immunized with pp65 vaccine. Thefirst and fourth plots show splenocyte response to stimulation withvaccinia virus VV/pp65 in culture, the fourth plot (FIG. 6D) being thenegative control of splenocytes from a mouse immunized with vectorcontrol alone. The second plot serves as a negative control in which thesplenocytes were not stimulated. The third plot is another negativecontrol of splenocytes from a pp65 vaccinated mouse; the splenocyteswere stimulated with recombinant vaccinia virus VV/gB expressingunrelated antigen gB in culture. Absence of response in the third plotsignifies a pp65-specific response in the first plot.

[0110] In FIGS. 6A-D, the x-axis represents presence of the cell surfacemarker CD8 on the surface of splenocytes, meaning that a dot to theright of the verticle line represents a splenocyte positive for CD8 onits surface (CD8 positive or CD8+), while a dot to the left of theverticle line represents a splenocyte that does not have CD8 on itssurface (CD8 negative or CD8−). The y-axis represents presence of IFN-γinside T cells, meaning that a dot above the horizontal line representsa functional splenocyte producing IFN-γ, while a dot below thehorizontal line represents a splenocyte not producing IFN-γ. The upperright quadrant represents splenocytes positive for both CD8 and IFN-γstaining, which are considered antigen-specific CD8+ lymphocytes.

[0111] In FIGS. 6A-D, the numbers in the upper right comer of each plotgive the ratio of dots occurring in the upper right quadrant relative tothe total dots in the right half of the plot. These double positivecells represent splenocytes that mount a specific CD8 T cell response tothe stimulation of related antigen pp65 expressed by the recombinantvaccinia virus VV/pp65 as they are not present in the unstimulated orstimulated with unrelated antigen plots. The presence of cells in theupper right quadrant in this first plot shows that the splenocytes froma mouse vaccinated with the pp65 vaccine were able to produce app65-specific response. The absence of the representation of cells inthe upper right quadrant in the third plot shows that a splenocyteresponse was not detectably produced in response to stimulation ofunrelated antigen gB expressed by recombinant vaccinia virus VV/gB,showing that vaccination with the pp65 DNA vaccine can inducesplenocytes that can specifically respond to pp65 stimulation, but notto gB stimulation.

[0112] The percentages of pp65-specific CD8⁺ T cells in splenocytes fromfive individual mice immunized with the pp65 vaccine are listed in Table2. These percentages were determined by FACS staining of cells andreflect percentages of CD8⁺ cells which stained positive for IFN-γ inresponse to stimulation with pp65 or non-specific (“unrelated”)antigens. As shown in Table 2, an average of 3.49 percent of totalsplenocytes from pp65-immunized mice were pp65-specfic CD8⁺ T cells.Percentages in individual mice ranged from 1.3 to 13.4 percent. In allmice, less than 0.1 percent of cells were specific for the unrelatedantigen. TABLE 2 CMV pp65 specific CD8⁺ T cell responses from miceimmunized with pp65 DNA vaccine (percentage of CD8⁺ T cells stainedpositive for IFN-γ secretion) stimulation 1 2 3 4 5 avg pp65 1.49 4.131.3 4.83 13.4 3.49 unrelated <0.1 <0.1 <0.1 <0.1 <0.1 <0.1

[0113] CD4⁺ T cell responses in individual mice were also determined.Table 3 lists the percentages of CD4⁺, pp65-specific (i.e.,IFN-γ-secreting) splenocytes as determined by FACS. An average of 0.71percent of splenocytes from immunized mice were pp65-specific CD4⁺ Tcells. Percentages in individual mice ranged from 0.31 to 3.99 percent.In all mice, less than 0.1 percent of cells were specific for theunrelated antigen. TABLE 3 CMV pp65 specific CD4+ T cell responses frommice immunized with pp65 DNA vaccine (percentage of CD4+ T cells stainedpositive for IFN-γ secretion). mouse 1 2 3 4 5 avg pp65 0.31 0.59 0.320.78 3.99 0.71 unrelated <0.1 <0.1 <0.1 <0.1 <0.1 <0.1

[0114] These data show that the pp65 vaccine induced a robustantigen-specific CD8⁺ and CD4⁺ T cell-mediated immune response in mice.

Example 5 pp150 Vaccine

[0115] Balb/C mice were immunized by gene gun three times at four-weekintervals with DNA vaccine expressing human CMV pp150. 6 μg of DNA wasadministered at each immunization.

[0116] At a point between 2 weeks and 6 months after the thirdimmunization of the mice, cell-mediated immunity was tested. On the dayof actual testing, the immunized mice were sacrificed, the spleen wasremoved, and splenocytes were isolated after red blood cell lysis.Splenocytes were then tested by specific stimulation with related HCMVantigen, namely recombinant vaccinia virus expressing pp150 (VV/pp150).

[0117] FIGS. 7A-D show the results of experiments in which mice wereimmunized with the pp150 vaccine. The results indicate that immunizedmice were capable of mounting a specific cell-mediated response tovaccinia virus VV/pp150 stimulation. The first three plots (FIGS. 7A-C)are results of a FACS analysis of splenocytes from a mouse immunizedwith the pp150 DNA vaccine. The first and fourth plots show splenocyteresponse to stimulation with vaccinia virus VV/pp150, the fourth plot(FIG. 7D) being the negative control. The second plot serves as anegative control in which the splenocytes were not stimulated. The thirdplot is another negative control of splenocytes from a pp150 vaccinatedmouse. The splenocytes in the third plot were stimulated withrecombinant vaccinia virus VV/gB expressing unrelated antigen gB.Absence of response in the third plot signifies a pp150-specificresponse in the first plot.

[0118] The x-axis of FIGS. 7A-D represents the presence of the cellsurface marker CD8 on the surface of splenocytes, meaning that a dot tothe right of the verticle line represents a splenocyte positive for CD8on its surface (CD8 positive or CD8+) while a dot to the left of theverticle line represents a cell that does not have CD8 on its surface(CD8 negative or CD8−). The y-axis represents the presence of IFN-γinside T cells, meaning that a dot above the horizontal line representsa functional splenocyte producing IFN-γ while a dot below the horizontalline represents a splenocytes not producing IFN-γ. Dots in the upperright hand quadrant represent splenocytes positive for both CD8 andIFN-γ staining, which are considered as antigen-specific CD8+lymphocytes.

[0119] In FIGS. 7A-D, the numbers in the upper right corner of each plotgive the ratio of dots occurring in the upper right quadrant relative tothe total dots in the right half of the plot. These double positivecells represent splenocytes that mount a specific CD8 T cell response tothe stimulation of related antigen expressed by the recombinant vacciniavirus VV/pp150 as they are not present in the unstimulated or stimulatedwith unrelated antigen plots. The presence of cells in the upper rightquadrant in this first-plot shows that the pp150 vaccine was able toinduce splenocytes that could respond specifically to a pp150stimulation. The absence of representation of cells in the upper rightquadrant in the third plot shows that a splenocyte response was notdetectably produced in response to stimulation of unrelated antigen gBexpressed by recombinant vaccinia virus VV/gB, showing that vaccinationwith the pp150 DNA vaccine can induce splenocytes that respondspecifically to pp150 stimulation, but not to gB stimulation.

[0120] The percentages of pp150-specific CD8⁺ T cells in splenocytesfrom 5 individual mice immunized with the pp150 vaccine are listed inTable 4. These percentages were determined by FACS staining of cells andreflect percentages of CD8⁺ cells which stained positive for IFN-γ inresponse to stimulation with pp150 or non-specific (“unrelated”)antigens. As shown, an average of 5.12 percent of total splenocytes frompp150-immunized mice were pp150-specfic CD8⁺ T cells, with percentagesin individual mice ranging from 1.05 to 13.4 percent. In all mice, lessthan 0.1 percent of cells were specific for the unrelated antigen. TABLE4 CMV pp150 specific CD8⁺ T cell responses from mice immunized withpp150 DNA vaccine stimulation 1 2 3 4 5 avg pp150 4.16 3.68 1.05 16.313.4 5.12 Unrelated antigen <0.1 <0.1 <0.1 <0.1 <0.1 <0.1

[0121] These data show that the pp150 vaccine induced a robustantigen-specific CD8⁺ T cell mediated response in mice.

Example 6 gH, gL, gO, and gcIII Vaccines

[0122] CMV gH, gL, and gO genes were cloned into the pJW4303 optimizedvaccine vector. The gH, gL, and gO polypeptides are depictedschematically in FIG. 8A. gH is the wild type glycoprotein H, which is743 amino acids long. gL is the wild type glycoprotein L, which is 278amino acids in length. gO is the wild type glycoprotein O, which is 466amino acids in length.

[0123] DNA vaccine vectors encoding each of these were transientlytransfected into 293T cells in the following combinations: gH alone,gH+gL, gH+gL+gO, gH+gL+gB, gH+gL+gO+gB. Lysates of cell transfectantswere analyzed for expression of gH and gL by western blotting. As shownin FIG. 8B, all of the transfectants into which a gH-expressing vectorwas introduced exhibited expression of gH. As shown in FIG. 8C, all ofthe transfectants into which a gL-expressing vector was introducedexhibited expression of gL. Positive control samples (i.e., CMV-infectedcells, lane 1 of FIG. 8B and FIG. 8C) showed expression of gH and gL.Negative control samples (uninfected cells, lane 2 of FIG. 8B and FIG.8C; and 293T cell transfected with empty vector, lane 8 of FIG. 8B andFIG. 8C) did not exhibit expression of gH or gL. These data show that gHand gL can be expressed from DNA vaccine vectors in mammalian cells.Previous studies suggested that co-expression of gL facilitated properprocessing and trafficking of gH through the secretory pathway (Spaeteet al., Virology, 193(2):853-861, 1993). We observed expression of each,without dependence on co-expression of the other, in contrast to ourobservation with gM and gN. However, we analyzed secreted andcell-associated forms of each protein by western blot. Cell lysate (“L”)and cell supernatants (“S”) of gH/L/O, gH/gL, gH/O, gH, and empty vectortransfectants were resolved by SDS-PAGE and blotted for gH and gLexpression. As shown in FIG. 9, upper panel, co-expression of gH and gLallows secretion of a form of gH, which was recovered from cellsupernatants (FIG. 9, upper panel, compare lane 4 to lanes 6 and 8).These data indicate that co-administration of gH and gL DNA vaccines isnot required for efficient expression of each gene product. However,co-expression may facilitate the secretion of gH.

Other Embodiments

[0124] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1 16 1 2721 DNA Human cytomegalovirus CDS (1)...(2718) 1 atg gaa tcc aggatc tgg tgc ctg gta gtc tgc gtt aac ctg tgt atc 48 Met Glu Ser Arg IleTrp Cys Leu Val Val Cys Val Asn Leu Cys Ile 1 5 10 15 gtc tgt ctg ggtgct gcg gtt tcc tct tct agt act tcc cat gca act 96 Val Cys Leu Gly AlaAla Val Ser Ser Ser Ser Thr Ser His Ala Thr 20 25 30 tct tct act cac aatgga agc cat act tct cgt acg acg tct gct caa 144 Ser Ser Thr His Asn GlySer His Thr Ser Arg Thr Thr Ser Ala Gln 35 40 45 acc cgg tca gtc tat tctcaa cac gta acg tct tct gaa gcc gtc agt 192 Thr Arg Ser Val Tyr Ser GlnHis Val Thr Ser Ser Glu Ala Val Ser 50 55 60 cat aga gcc aac gag act atctac aac act acc ctc aag tac gga gat 240 His Arg Ala Asn Glu Thr Ile TyrAsn Thr Thr Leu Lys Tyr Gly Asp 65 70 75 80 gtg gtg gga gtc aac act accaag tac ccc tat cgc gtg tgt tct atg 288 Val Val Gly Val Asn Thr Thr LysTyr Pro Tyr Arg Val Cys Ser Met 85 90 95 gcc cag ggt acg gat ctt att cgcttt gaa cgt aat atc atc tgc acc 336 Ala Gln Gly Thr Asp Leu Ile Arg PheGlu Arg Asn Ile Ile Cys Thr 100 105 110 tcg atg aag cct atc aat gaa gacttg gat gag ggc atc atg gtg gtc 384 Ser Met Lys Pro Ile Asn Glu Asp LeuAsp Glu Gly Ile Met Val Val 115 120 125 tac aag cgc aac atc gtg gcg cacacc ttt aag gta cgg gtc tac caa 432 Tyr Lys Arg Asn Ile Val Ala His ThrPhe Lys Val Arg Val Tyr Gln 130 135 140 aag gtt ttg acg ttt cgt cgt agctac gct tac atc tac acc act tat 480 Lys Val Leu Thr Phe Arg Arg Ser TyrAla Tyr Ile Tyr Thr Thr Tyr 145 150 155 160 ctg ctg ggc agc aat acg gaatac gtg gcg cct cct atg tgg gag att 528 Leu Leu Gly Ser Asn Thr Glu TyrVal Ala Pro Pro Met Trp Glu Ile 165 170 175 cat cac atc aac aag ttt gctcaa tgc tac agt tcc tac agc cgc gtt 576 His His Ile Asn Lys Phe Ala GlnCys Tyr Ser Ser Tyr Ser Arg Val 180 185 190 ata gga ggc acg gtt ttc gtggca tat cat agg gac agt tat gaa aac 624 Ile Gly Gly Thr Val Phe Val AlaTyr His Arg Asp Ser Tyr Glu Asn 195 200 205 aaa acc atg caa tta att cccgac gat tat tcc aac acc cac agt acc 672 Lys Thr Met Gln Leu Ile Pro AspAsp Tyr Ser Asn Thr His Ser Thr 210 215 220 cgt tac gtg acg gtc aag gatcag tgg cac agc cgc ggc agc acc tgg 720 Arg Tyr Val Thr Val Lys Asp GlnTrp His Ser Arg Gly Ser Thr Trp 225 230 235 240 ctc tat cgt gag acc tgtaat ctg aac tgt atg ctg acc atc act act 768 Leu Tyr Arg Glu Thr Cys AsnLeu Asn Cys Met Leu Thr Ile Thr Thr 245 250 255 gcg cgc tcc aag tat ccttat cat ttt ttt gca act tcc acg ggt gat 816 Ala Arg Ser Lys Tyr Pro TyrHis Phe Phe Ala Thr Ser Thr Gly Asp 260 265 270 gtg gtt tac att tct cctttc tac aac gga acc aat cgc aat gcc agc 864 Val Val Tyr Ile Ser Pro PheTyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285 tac ttt gga gaa aac gccgac aag ttt ttc att ttc ccg aac tac acc 912 Tyr Phe Gly Glu Asn Ala AspLys Phe Phe Ile Phe Pro Asn Tyr Thr 290 295 300 atc gtt tcc gac ttt ggaaga ccc aac gct gcg cca gaa acc cat agg 960 Ile Val Ser Asp Phe Gly ArgPro Asn Ala Ala Pro Glu Thr His Arg 305 310 315 320 ttg gtg gct ttt ctcgaa cgt gcc gac tcg gtg atc tct tgg gat ata 1008 Leu Val Ala Phe Leu GluArg Ala Asp Ser Val Ile Ser Trp Asp Ile 325 330 335 cag gac gag aag aatgtc acc tgc cag ctc acc ttc tgg gaa gcc tcg 1056 Gln Asp Glu Lys Asn ValThr Cys Gln Leu Thr Phe Trp Glu Ala Ser 340 345 350 gaa cgt act atc cgttcc gaa gcc gaa gac tcg tac cac ttt tct tct 1104 Glu Arg Thr Ile Arg SerGlu Ala Glu Asp Ser Tyr His Phe Ser Ser 355 360 365 gcc aaa atg act gcaact ttt ctg tct aag aaa caa gaa gtg aac atg 1152 Ala Lys Met Thr Ala ThrPhe Leu Ser Lys Lys Gln Glu Val Asn Met 370 375 380 tcc gac tcc gcg ctggac tgc gta cgt gat gag gct ata aat aag tta 1200 Ser Asp Ser Ala Leu AspCys Val Arg Asp Glu Ala Ile Asn Lys Leu 385 390 395 400 cag cag att ttcaat act tca tac aat caa aca tat gaa aaa tac gga 1248 Gln Gln Ile Phe AsnThr Ser Tyr Asn Gln Thr Tyr Glu Lys Tyr Gly 405 410 415 aac gtg tcc gtcttc gaa acc agc ggc ggt ctg gtg gtg ttc tgg caa 1296 Asn Val Ser Val PheGlu Thr Ser Gly Gly Leu Val Val Phe Trp Gln 420 425 430 ggc atc aag caaaaa tct ttg gtg gaa ttg gaa cgt ttg gcc aat cga 1344 Gly Ile Lys Gln LysSer Leu Val Glu Leu Glu Arg Leu Ala Asn Arg 435 440 445 tcc agt ctg aatatc act cat agg acc aga aga agt acg agt gac aat 1392 Ser Ser Leu Asn IleThr His Arg Thr Arg Arg Ser Thr Ser Asp Asn 450 455 460 aat aca act catttg tcc agc atg gaa tcg gtg cac aat ctg gtc tac 1440 Asn Thr Thr His LeuSer Ser Met Glu Ser Val His Asn Leu Val Tyr 465 470 475 480 gcc cag ctgcag ttc acc tat gac acg ttg cgc ggt tac atc aac cgg 1488 Ala Gln Leu GlnPhe Thr Tyr Asp Thr Leu Arg Gly Tyr Ile Asn Arg 485 490 495 gcg ctg gcgcaa atc gca gaa gcc tgg tgt gtg gat caa cgg cgc acc 1536 Ala Leu Ala GlnIle Ala Glu Ala Trp Cys Val Asp Gln Arg Arg Thr 500 505 510 cta gag gtcttc aag gaa ctc agc aag atc aac ccg tca gcc att ctc 1584 Leu Glu Val PheLys Glu Leu Ser Lys Ile Asn Pro Ser Ala Ile Leu 515 520 525 tcg gcc atttac aac aaa ccg att gcc gcg cgt ttc atg ggt gat gtc 1632 Ser Ala Ile TyrAsn Lys Pro Ile Ala Ala Arg Phe Met Gly Asp Val 530 535 540 ttg ggc ctggcc agc tgc gtg acc atc aac caa acc agc gtc aag gtg 1680 Leu Gly Leu AlaSer Cys Val Thr Ile Asn Gln Thr Ser Val Lys Val 545 550 555 560 ctg cgtgat atg aac gtg aag gaa tcg cca gga cgc tgc tac tca cga 1728 Leu Arg AspMet Asn Val Lys Glu Ser Pro Gly Arg Cys Tyr Ser Arg 565 570 575 ccc gtggtc atc ttt aat ttc gcc aac agc tcg tac gtg cag tac ggt 1776 Pro Val ValIle Phe Asn Phe Ala Asn Ser Ser Tyr Val Gln Tyr Gly 580 585 590 caa ctgggc gag gac aac gaa atc ctg ttg ggc aac cac cgc act gag 1824 Gln Leu GlyGlu Asp Asn Glu Ile Leu Leu Gly Asn His Arg Thr Glu 595 600 605 gaa tgtcag ctt ccc agc ctc aag atc ttc atc gcc ggg aac tcg gcc 1872 Glu Cys GlnLeu Pro Ser Leu Lys Ile Phe Ile Ala Gly Asn Ser Ala 610 615 620 tac gagtac gtg gac tac ctc ttc aaa cgc atg att gac ctc agc agt 1920 Tyr Glu TyrVal Asp Tyr Leu Phe Lys Arg Met Ile Asp Leu Ser Ser 625 630 635 640 atctcc acc gtc gac agc atg atc gcc ctg gat atc gac ccg ctg gaa 1968 Ile SerThr Val Asp Ser Met Ile Ala Leu Asp Ile Asp Pro Leu Glu 645 650 655 aatacc gac ttc agg gta ctg gaa ctt tac tcg cag aaa gag ctg cgt 2016 Asn ThrAsp Phe Arg Val Leu Glu Leu Tyr Ser Gln Lys Glu Leu Arg 660 665 670 tccagc aac gtt ttt gac ctc gaa gag atc atg cgc gaa ttc aac tcg 2064 Ser SerAsn Val Phe Asp Leu Glu Glu Ile Met Arg Glu Phe Asn Ser 675 680 685 tacaag cag cgg gta aag tac gtg gag gac aag gta gtc gac ccg cta 2112 Tyr LysGln Arg Val Lys Tyr Val Glu Asp Lys Val Val Asp Pro Leu 690 695 700 ccgccc tac ctc aag ggt ctg gac gac ctc atg agc ggc ctg ggc gcc 2160 Pro ProTyr Leu Lys Gly Leu Asp Asp Leu Met Ser Gly Leu Gly Ala 705 710 715 720gcg gga aag gcc gtt ggc gta gcc att ggg gcc gtg ggt ggc gcg gtg 2208 AlaGly Lys Ala Val Gly Val Ala Ile Gly Ala Val Gly Gly Ala Val 725 730 735gcc tcc gtg gtc gaa ggc gtt gcc acc ttc ctc aaa aac ccc ttc gga 2256 AlaSer Val Val Glu Gly Val Ala Thr Phe Leu Lys Asn Pro Phe Gly 740 745 750gcc ttc acc atc atc ctc gtg gcc ata gcc gta gtc att atc act tat 2304 AlaPhe Thr Ile Ile Leu Val Ala Ile Ala Val Val Ile Ile Thr Tyr 755 760 765ttg atc tat act cga cag cgg cgt ctg tgc acg cag ccg ctg cag aac 2352 LeuIle Tyr Thr Arg Gln Arg Arg Leu Cys Thr Gln Pro Leu Gln Asn 770 775 780ctc ttt ccc tat ctg gtg tcc gcc gac ggg acc acc gtg acg tcg ggc 2400 LeuPhe Pro Tyr Leu Val Ser Ala Asp Gly Thr Thr Val Thr Ser Gly 785 790 795800 agc acc aaa gac acg tcg tta cag gct ccg cct tcc tac gag gaa agt 2448Ser Thr Lys Asp Thr Ser Leu Gln Ala Pro Pro Ser Tyr Glu Glu Ser 805 810815 gtt tat aat tct ggt cgc aaa gga ccg gga cca ccg tcg tct gat gca 2496Val Tyr Asn Ser Gly Arg Lys Gly Pro Gly Pro Pro Ser Ser Asp Ala 820 825830 tcc acg gcg gct ccg cct tac acc aac gag cag gct tac cag atg ctt 2544Ser Thr Ala Ala Pro Pro Tyr Thr Asn Glu Gln Ala Tyr Gln Met Leu 835 840845 ctg gcc ctg gcc cgt ctg gac gca gag cag cga gcg cag cag aac ggt 2592Leu Ala Leu Ala Arg Leu Asp Ala Glu Gln Arg Ala Gln Gln Asn Gly 850 855860 aca gat tct ttg gac gga cag act ggc acg cag gac aag gga cag aag 2640Thr Asp Ser Leu Asp Gly Gln Thr Gly Thr Gln Asp Lys Gly Gln Lys 865 870875 880 cct aac ctg cta gac cgg ctg cga cat cgc aaa aac ggc tac aga cac2688 Pro Asn Leu Leu Asp Arg Leu Arg His Arg Lys Asn Gly Tyr Arg His 885890 895 ttg aaa gac tcc gac gaa gaa gag aac gtc tga 2721 Leu Lys Asp SerAsp Glu Glu Glu Asn Val 900 905 2 906 PRT Human cytomegalovirus 2 MetGlu Ser Arg Ile Trp Cys Leu Val Val Cys Val Asn Leu Cys Ile 1 5 10 15Val Cys Leu Gly Ala Ala Val Ser Ser Ser Ser Thr Ser His Ala Thr 20 25 30Ser Ser Thr His Asn Gly Ser His Thr Ser Arg Thr Thr Ser Ala Gln 35 40 45Thr Arg Ser Val Tyr Ser Gln His Val Thr Ser Ser Glu Ala Val Ser 50 55 60His Arg Ala Asn Glu Thr Ile Tyr Asn Thr Thr Leu Lys Tyr Gly Asp 65 70 7580 Val Val Gly Val Asn Thr Thr Lys Tyr Pro Tyr Arg Val Cys Ser Met 85 9095 Ala Gln Gly Thr Asp Leu Ile Arg Phe Glu Arg Asn Ile Ile Cys Thr 100105 110 Ser Met Lys Pro Ile Asn Glu Asp Leu Asp Glu Gly Ile Met Val Val115 120 125 Tyr Lys Arg Asn Ile Val Ala His Thr Phe Lys Val Arg Val TyrGln 130 135 140 Lys Val Leu Thr Phe Arg Arg Ser Tyr Ala Tyr Ile Tyr ThrThr Tyr 145 150 155 160 Leu Leu Gly Ser Asn Thr Glu Tyr Val Ala Pro ProMet Trp Glu Ile 165 170 175 His His Ile Asn Lys Phe Ala Gln Cys Tyr SerSer Tyr Ser Arg Val 180 185 190 Ile Gly Gly Thr Val Phe Val Ala Tyr HisArg Asp Ser Tyr Glu Asn 195 200 205 Lys Thr Met Gln Leu Ile Pro Asp AspTyr Ser Asn Thr His Ser Thr 210 215 220 Arg Tyr Val Thr Val Lys Asp GlnTrp His Ser Arg Gly Ser Thr Trp 225 230 235 240 Leu Tyr Arg Glu Thr CysAsn Leu Asn Cys Met Leu Thr Ile Thr Thr 245 250 255 Ala Arg Ser Lys TyrPro Tyr His Phe Phe Ala Thr Ser Thr Gly Asp 260 265 270 Val Val Tyr IleSer Pro Phe Tyr Asn Gly Thr Asn Arg Asn Ala Ser 275 280 285 Tyr Phe GlyGlu Asn Ala Asp Lys Phe Phe Ile Phe Pro Asn Tyr Thr 290 295 300 Ile ValSer Asp Phe Gly Arg Pro Asn Ala Ala Pro Glu Thr His Arg 305 310 315 320Leu Val Ala Phe Leu Glu Arg Ala Asp Ser Val Ile Ser Trp Asp Ile 325 330335 Gln Asp Glu Lys Asn Val Thr Cys Gln Leu Thr Phe Trp Glu Ala Ser 340345 350 Glu Arg Thr Ile Arg Ser Glu Ala Glu Asp Ser Tyr His Phe Ser Ser355 360 365 Ala Lys Met Thr Ala Thr Phe Leu Ser Lys Lys Gln Glu Val AsnMet 370 375 380 Ser Asp Ser Ala Leu Asp Cys Val Arg Asp Glu Ala Ile AsnLys Leu 385 390 395 400 Gln Gln Ile Phe Asn Thr Ser Tyr Asn Gln Thr TyrGlu Lys Tyr Gly 405 410 415 Asn Val Ser Val Phe Glu Thr Ser Gly Gly LeuVal Val Phe Trp Gln 420 425 430 Gly Ile Lys Gln Lys Ser Leu Val Glu LeuGlu Arg Leu Ala Asn Arg 435 440 445 Ser Ser Leu Asn Ile Thr His Arg ThrArg Arg Ser Thr Ser Asp Asn 450 455 460 Asn Thr Thr His Leu Ser Ser MetGlu Ser Val His Asn Leu Val Tyr 465 470 475 480 Ala Gln Leu Gln Phe ThrTyr Asp Thr Leu Arg Gly Tyr Ile Asn Arg 485 490 495 Ala Leu Ala Gln IleAla Glu Ala Trp Cys Val Asp Gln Arg Arg Thr 500 505 510 Leu Glu Val PheLys Glu Leu Ser Lys Ile Asn Pro Ser Ala Ile Leu 515 520 525 Ser Ala IleTyr Asn Lys Pro Ile Ala Ala Arg Phe Met Gly Asp Val 530 535 540 Leu GlyLeu Ala Ser Cys Val Thr Ile Asn Gln Thr Ser Val Lys Val 545 550 555 560Leu Arg Asp Met Asn Val Lys Glu Ser Pro Gly Arg Cys Tyr Ser Arg 565 570575 Pro Val Val Ile Phe Asn Phe Ala Asn Ser Ser Tyr Val Gln Tyr Gly 580585 590 Gln Leu Gly Glu Asp Asn Glu Ile Leu Leu Gly Asn His Arg Thr Glu595 600 605 Glu Cys Gln Leu Pro Ser Leu Lys Ile Phe Ile Ala Gly Asn SerAla 610 615 620 Tyr Glu Tyr Val Asp Tyr Leu Phe Lys Arg Met Ile Asp LeuSer Ser 625 630 635 640 Ile Ser Thr Val Asp Ser Met Ile Ala Leu Asp IleAsp Pro Leu Glu 645 650 655 Asn Thr Asp Phe Arg Val Leu Glu Leu Tyr SerGln Lys Glu Leu Arg 660 665 670 Ser Ser Asn Val Phe Asp Leu Glu Glu IleMet Arg Glu Phe Asn Ser 675 680 685 Tyr Lys Gln Arg Val Lys Tyr Val GluAsp Lys Val Val Asp Pro Leu 690 695 700 Pro Pro Tyr Leu Lys Gly Leu AspAsp Leu Met Ser Gly Leu Gly Ala 705 710 715 720 Ala Gly Lys Ala Val GlyVal Ala Ile Gly Ala Val Gly Gly Ala Val 725 730 735 Ala Ser Val Val GluGly Val Ala Thr Phe Leu Lys Asn Pro Phe Gly 740 745 750 Ala Phe Thr IleIle Leu Val Ala Ile Ala Val Val Ile Ile Thr Tyr 755 760 765 Leu Ile TyrThr Arg Gln Arg Arg Leu Cys Thr Gln Pro Leu Gln Asn 770 775 780 Leu PhePro Tyr Leu Val Ser Ala Asp Gly Thr Thr Val Thr Ser Gly 785 790 795 800Ser Thr Lys Asp Thr Ser Leu Gln Ala Pro Pro Ser Tyr Glu Glu Ser 805 810815 Val Tyr Asn Ser Gly Arg Lys Gly Pro Gly Pro Pro Ser Ser Asp Ala 820825 830 Ser Thr Ala Ala Pro Pro Tyr Thr Asn Glu Gln Ala Tyr Gln Met Leu835 840 845 Leu Ala Leu Ala Arg Leu Asp Ala Glu Gln Arg Ala Gln Gln AsnGly 850 855 860 Thr Asp Ser Leu Asp Gly Gln Thr Gly Thr Gln Asp Lys GlyGln Lys 865 870 875 880 Pro Asn Leu Leu Asp Arg Leu Arg His Arg Lys AsnGly Tyr Arg His 885 890 895 Leu Lys Asp Ser Asp Glu Glu Glu Asn Val 900905 3 1119 DNA Human cytomegalovirus CDS (1)...(1116) 3 atg gcc ccc tcgcac gtg gat aag gtg aat aca cgg aca tgg agc gct 48 Met Ala Pro Ser HisVal Asp Lys Val Asn Thr Arg Thr Trp Ser Ala 1 5 10 15 tct atc gtt ttcatg gtg ctg act ttt gtc aac gtc agc gtg cat cta 96 Ser Ile Val Phe MetVal Leu Thr Phe Val Asn Val Ser Val His Leu 20 25 30 gtg ctg agc aat tttccg cac ctg ggc tac ccc tgc gtc tac tat cac 144 Val Leu Ser Asn Phe ProHis Leu Gly Tyr Pro Cys Val Tyr Tyr His 35 40 45 gtc gtg gac ttt gaa aggctc aac atg tcg gcc tac aac gta atg cac 192 Val Val Asp Phe Glu Arg LeuAsn Met Ser Ala Tyr Asn Val Met His 50 55 60 ctg cac acg cct atg ctt ttctta gac tcg gtg cag ttg gtg tgc tac 240 Leu His Thr Pro Met Leu Phe LeuAsp Ser Val Gln Leu Val Cys Tyr 65 70 75 80 gcc gtg ttc atg cag ctc gtcttt tta gcc gtg acc atc tac tac ctg 288 Ala Val Phe Met Gln Leu Val PheLeu Ala Val Thr Ile Tyr Tyr Leu 85 90 95 gta tgc tgg atc aag atc agc atgcgc aag gac aaa ggc atg agc cta 336 Val Cys Trp Ile Lys Ile Ser Met ArgLys Asp Lys Gly Met Ser Leu 100 105 110 aac cag tcg aca cgc gac att tcgtac atg ggc gac agc ctc aca gcc 384 Asn Gln Ser Thr Arg Asp Ile Ser TyrMet Gly Asp Ser Leu Thr Ala 115 120 125 ttc ctc ttc att ctc agc atg gacacg ttc caa cta ttc aca ctg acc 432 Phe Leu Phe Ile Leu Ser Met Asp ThrPhe Gln Leu Phe Thr Leu Thr 130 135 140 atg tca ttt cgg ctg ccc agc atgatc gcc ttc atg gcc gcc gtg cac 480 Met Ser Phe Arg Leu Pro Ser Met IleAla Phe Met Ala Ala Val His 145 150 155 160 ttt ttc tgc ctg acc att ttcaac gtg agc atg gtc acg cag tac cgc 528 Phe Phe Cys Leu Thr Ile Phe AsnVal Ser Met Val Thr Gln Tyr Arg 165 170 175 agc tac aaa cgc tca ctc tttttc ttc tcg cgt ctg cac ccc aag ctc 576 Ser Tyr Lys Arg Ser Leu Phe PhePhe Ser Arg Leu His Pro Lys Leu 180 185 190 aaa ggt acg gtg cag ttc cgcacg ctc atc gtc aac ctg gta gag gta 624 Lys Gly Thr Val Gln Phe Arg ThrLeu Ile Val Asn Leu Val Glu Val 195 200 205 gcg ctt ggt ttc aac acc accgtg gta gcc atg gcc ctg tgc tac ggc 672 Ala Leu Gly Phe Asn Thr Thr ValVal Ala Met Ala Leu Cys Tyr Gly 210 215 220 ttc gga aac aac ttt ttc gtgcgt aca ggc cac atg gtg tta gcc gtc 720 Phe Gly Asn Asn Phe Phe Val ArgThr Gly His Met Val Leu Ala Val 225 230 235 240 ttc gtg gtc tac gct atcatc tcc atc atc tac ttt tta ctg atc gag 768 Phe Val Val Tyr Ala Ile IleSer Ile Ile Tyr Phe Leu Leu Ile Glu 245 250 255 gcc gtc ttt ttt caa tacgtc aag gtg caa ttc ggc tac cac ctg ggc 816 Ala Val Phe Phe Gln Tyr ValLys Val Gln Phe Gly Tyr His Leu Gly 260 265 270 gcc ttc ttt gga ctc tgcggc ctc atc tac ccc atc gtg cag tac gat 864 Ala Phe Phe Gly Leu Cys GlyLeu Ile Tyr Pro Ile Val Gln Tyr Asp 275 280 285 acc ttc ctc agc aac gaatac cgc acc ggc atc agc tgg tcg ttc ggc 912 Thr Phe Leu Ser Asn Glu TyrArg Thr Gly Ile Ser Trp Ser Phe Gly 290 295 300 atg ctc ttt ttc ata tgggcc atg ttt acg acg tgt cgc gcc gtc cgc 960 Met Leu Phe Phe Ile Trp AlaMet Phe Thr Thr Cys Arg Ala Val Arg 305 310 315 320 tac ttt cgc gga cgcggt agc ggc agt gtc aag tac cag gcg ctg gcc 1008 Tyr Phe Arg Gly Arg GlySer Gly Ser Val Lys Tyr Gln Ala Leu Ala 325 330 335 aca gcc tcc ggc gaagaa gtc gct gtg ctc agt cac cac gac agc ttg 1056 Thr Ala Ser Gly Glu GluVal Ala Val Leu Ser His His Asp Ser Leu 340 345 350 gaa agc cgt cgc ctccgc gaa gaa gag gac gac gac gat gat gaa gac 1104 Glu Ser Arg Arg Leu ArgGlu Glu Glu Asp Asp Asp Asp Asp Glu Asp 355 360 365 ttc gag gac gct taa1119 Phe Glu Asp Ala 370 4 372 PRT Human cytomegalovirus 4 Met Ala ProSer His Val Asp Lys Val Asn Thr Arg Thr Trp Ser Ala 1 5 10 15 Ser IleVal Phe Met Val Leu Thr Phe Val Asn Val Ser Val His Leu 20 25 30 Val LeuSer Asn Phe Pro His Leu Gly Tyr Pro Cys Val Tyr Tyr His 35 40 45 Val ValAsp Phe Glu Arg Leu Asn Met Ser Ala Tyr Asn Val Met His 50 55 60 Leu HisThr Pro Met Leu Phe Leu Asp Ser Val Gln Leu Val Cys Tyr 65 70 75 80 AlaVal Phe Met Gln Leu Val Phe Leu Ala Val Thr Ile Tyr Tyr Leu 85 90 95 ValCys Trp Ile Lys Ile Ser Met Arg Lys Asp Lys Gly Met Ser Leu 100 105 110Asn Gln Ser Thr Arg Asp Ile Ser Tyr Met Gly Asp Ser Leu Thr Ala 115 120125 Phe Leu Phe Ile Leu Ser Met Asp Thr Phe Gln Leu Phe Thr Leu Thr 130135 140 Met Ser Phe Arg Leu Pro Ser Met Ile Ala Phe Met Ala Ala Val His145 150 155 160 Phe Phe Cys Leu Thr Ile Phe Asn Val Ser Met Val Thr GlnTyr Arg 165 170 175 Ser Tyr Lys Arg Ser Leu Phe Phe Phe Ser Arg Leu HisPro Lys Leu 180 185 190 Lys Gly Thr Val Gln Phe Arg Thr Leu Ile Val AsnLeu Val Glu Val 195 200 205 Ala Leu Gly Phe Asn Thr Thr Val Val Ala MetAla Leu Cys Tyr Gly 210 215 220 Phe Gly Asn Asn Phe Phe Val Arg Thr GlyHis Met Val Leu Ala Val 225 230 235 240 Phe Val Val Tyr Ala Ile Ile SerIle Ile Tyr Phe Leu Leu Ile Glu 245 250 255 Ala Val Phe Phe Gln Tyr ValLys Val Gln Phe Gly Tyr His Leu Gly 260 265 270 Ala Phe Phe Gly Leu CysGly Leu Ile Tyr Pro Ile Val Gln Tyr Asp 275 280 285 Thr Phe Leu Ser AsnGlu Tyr Arg Thr Gly Ile Ser Trp Ser Phe Gly 290 295 300 Met Leu Phe PheIle Trp Ala Met Phe Thr Thr Cys Arg Ala Val Arg 305 310 315 320 Tyr PheArg Gly Arg Gly Ser Gly Ser Val Lys Tyr Gln Ala Leu Ala 325 330 335 ThrAla Ser Gly Glu Glu Val Ala Val Leu Ser His His Asp Ser Leu 340 345 350Glu Ser Arg Arg Leu Arg Glu Glu Glu Asp Asp Asp Asp Asp Glu Asp 355 360365 Phe Glu Asp Ala 370 5 417 DNA Human cytomegalovirus CDS (1)...(414)5 atg gag tgg aac aca cta gta tta ggt ctt tta gtt tta tcg gta gtg 48 MetGlu Trp Asn Thr Leu Val Leu Gly Leu Leu Val Leu Ser Val Val 1 5 10 15gca gag agt tct ggt aac aat tca tcc acg tca acc tct gca act aca 96 AlaGlu Ser Ser Gly Asn Asn Ser Ser Thr Ser Thr Ser Ala Thr Thr 20 25 30 tcaaag tct tct gct agc gta tca act acc aaa cta aca aca gtt gca 144 Ser LysSer Ser Ala Ser Val Ser Thr Thr Lys Leu Thr Thr Val Ala 35 40 45 aca acttct gca aca act acg acg act acg acc tta tcg aca act agc 192 Thr Thr SerAla Thr Thr Thr Thr Thr Thr Thr Leu Ser Thr Thr Ser 50 55 60 act aaa ctcagt tct acc acc cac gat cct aat gtg atg aga cga cat 240 Thr Lys Leu SerSer Thr Thr His Asp Pro Asn Val Met Arg Arg His 65 70 75 80 gcg aac gatgat ttt tac aag gcg cat tgc aca tcg cat atg tat gag 288 Ala Asn Asp AspPhe Tyr Lys Ala His Cys Thr Ser His Met Tyr Glu 85 90 95 ctc tca ctg tccagc ttt gcg gcc tgg tgg act atg ctt aat gct cta 336 Leu Ser Leu Ser SerPhe Ala Ala Trp Trp Thr Met Leu Asn Ala Leu 100 105 110 att ctc atg ggagct ttt tgt att gta cta cga cat tgc tgc ttc cag 384 Ile Leu Met Gly AlaPhe Cys Ile Val Leu Arg His Cys Cys Phe Gln 115 120 125 aac ttt act gcaacc acc acc aaa ggc tat tga 417 Asn Phe Thr Ala Thr Thr Thr Lys Gly Tyr130 135 6 138 PRT Human cytomegalovirus 6 Met Glu Trp Asn Thr Leu ValLeu Gly Leu Leu Val Leu Ser Val Val 1 5 10 15 Ala Glu Ser Ser Gly AsnAsn Ser Ser Thr Ser Thr Ser Ala Thr Thr 20 25 30 Ser Lys Ser Ser Ala SerVal Ser Thr Thr Lys Leu Thr Thr Val Ala 35 40 45 Thr Thr Ser Ala Thr ThrThr Thr Thr Thr Thr Leu Ser Thr Thr Ser 50 55 60 Thr Lys Leu Ser Ser ThrThr His Asp Pro Asn Val Met Arg Arg His 65 70 75 80 Ala Asn Asp Asp PheTyr Lys Ala His Cys Thr Ser His Met Tyr Glu 85 90 95 Leu Ser Leu Ser SerPhe Ala Ala Trp Trp Thr Met Leu Asn Ala Leu 100 105 110 Ile Leu Met GlyAla Phe Cys Ile Val Leu Arg His Cys Cys Phe Gln 115 120 125 Asn Phe ThrAla Thr Thr Thr Lys Gly Tyr 130 135 7 2232 DNA Human cytomegalovirus CDS(1)...(2229) 7 atg cgg ccc ggc ctc ccc ccc tac ctc act gtc ttc acc gtctac ctc 48 Met Arg Pro Gly Leu Pro Pro Tyr Leu Thr Val Phe Thr Val TyrLeu 1 5 10 15 ctc agt cac cta cct tcg caa cga tat ggc gcg gac gcc gcatcc gaa 96 Leu Ser His Leu Pro Ser Gln Arg Tyr Gly Ala Asp Ala Ala SerGlu 20 25 30 gcg ctg gac cct cac gca ttt cac cta cta ctc aac acc tac gggaga 144 Ala Leu Asp Pro His Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg35 40 45 ccc atc cgc ttc ctg cgt gaa aac acc acc cag tgc acc tac aac agc192 Pro Ile Arg Phe Leu Arg Glu Asn Thr Thr Gln Cys Thr Tyr Asn Ser 5055 60 agc ctc cgt aac agc acg gtc gtc agg gaa aac gcc atc agt ttc aac240 Ser Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala Ile Ser Phe Asn 6570 75 80 ttt ttc caa agc tat aat caa tac tat gta ttc cat atg cct cga tgt288 Phe Phe Gln Ser Tyr Asn Gln Tyr Tyr Val Phe His Met Pro Arg Cys 8590 95 ctt ttt gcg ggt cct ctg gcg gag cag ttt ctg aac cag gta gat ctg336 Leu Phe Ala Gly Pro Leu Ala Glu Gln Phe Leu Asn Gln Val Asp Leu 100105 110 acc gaa acc cta gaa aga tac caa cag aga ctt aac acc tac gca ttg384 Thr Glu Thr Leu Glu Arg Tyr Gln Gln Arg Leu Asn Thr Tyr Ala Leu 115120 125 gta tcc aaa gac ctg gcc agc tac cga tct ttt tcg cag cag ctg aag432 Val Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gln Gln Leu Lys 130135 140 gca caa gac agc ctg ggt cag cag ccc acc acc gtg cca ccg ccc att480 Ala Gln Asp Ser Leu Gly Gln Gln Pro Thr Thr Val Pro Pro Pro Ile 145150 155 160 gat ctg tca ata cct cac gtt tgg atg cca ccc caa acc act ccacac 528 Asp Leu Ser Ile Pro His Val Trp Met Pro Pro Gln Thr Thr Pro His165 170 175 gac tgg aag gga tcg cac acc acc tcg gga cta cat cgg cca cacttt 576 Asp Trp Lys Gly Ser His Thr Thr Ser Gly Leu His Arg Pro His Phe180 185 190 aac cag acc tgt atc ctc ttt gat gga cac gat ctg ctt ttc agcacc 624 Asn Gln Thr Cys Ile Leu Phe Asp Gly His Asp Leu Leu Phe Ser Thr195 200 205 gtt acg ccc tgt ctg cac cag ggc ttt tac ctc atg gac gaa ctacgt 672 Val Thr Pro Cys Leu His Gln Gly Phe Tyr Leu Met Asp Glu Leu Arg210 215 220 tac gtt aaa atc aca ctg acc gag gac ttc ttc gta gtt acg gtatct 720 Tyr Val Lys Ile Thr Leu Thr Glu Asp Phe Phe Val Val Thr Val Ser225 230 235 240 ata gac gac gac aca ccc atg ctg ctt atc ttc ggt cat cttcca cgc 768 Ile Asp Asp Asp Thr Pro Met Leu Leu Ile Phe Gly His Leu ProArg 245 250 255 gta ctc ttc aaa gcg ccc tat caa cgc gac aac ttt ata ctacga caa 816 Val Leu Phe Lys Ala Pro Tyr Gln Arg Asp Asn Phe Ile Leu ArgGln 260 265 270 act gaa aaa cac gag ctc ctg gta cta gtt aag aaa gct caacta aac 864 Thr Glu Lys His Glu Leu Leu Val Leu Val Lys Lys Ala Gln LeuAsn 275 280 285 cgt cac tcc tat ctc aaa gac tcg gac ttt ctc gac gcc gcactc gac 912 Arg His Ser Tyr Leu Lys Asp Ser Asp Phe Leu Asp Ala Ala LeuAsp 290 295 300 ttc aac tac ctg gac ctc agc gca ctg tta cgt aac agc tttcac cgt 960 Phe Asn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe HisArg 305 310 315 320 tac gct gta gac gta ctc aaa agc ggt cga tgt caa atgttg gac cgc 1008 Tyr Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gln Met LeuAsp Arg 325 330 335 cgc acg gta gaa atg gcc ttc gcc tac gca tta gca ctgttc gcg gca 1056 Arg Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu PheAla Ala 340 345 350 gcc cga caa gaa gag gcc ggc acc gaa atc tcc atc ccacga gcc cta 1104 Ala Arg Gln Glu Glu Ala Gly Thr Glu Ile Ser Ile Pro ArgAla Leu 355 360 365 gac cgc cag gcc gca ctc tta caa ata caa gaa ttt atgatc acc tgc 1152 Asp Arg Gln Ala Ala Leu Leu Gln Ile Gln Glu Phe Met IleThr Cys 370 375 380 ctc tca caa aca cca cca cgc acc aca ttg ctg cta tatccc aca gcc 1200 Leu Ser Gln Thr Pro Pro Arg Thr Thr Leu Leu Leu Tyr ProThr Ala 385 390 395 400 gtg gac ctg gcc aaa cga gcc ctc tgg acg ccg gaccag atc acc gac 1248 Val Asp Leu Ala Lys Arg Ala Leu Trp Thr Pro Asp GlnIle Thr Asp 405 410 415 atc acc agc ctc gta cgc ctg gtc tac ata ctt tctaaa cag aat cag 1296 Ile Thr Ser Leu Val Arg Leu Val Tyr Ile Leu Ser LysGln Asn Gln 420 425 430 caa cat ctc att ccc cag tgg gca cta cga cag atcgcc gac ttt gcc 1344 Gln His Leu Ile Pro Gln Trp Ala Leu Arg Gln Ile AlaAsp Phe Ala 435 440 445 cta caa tta cac aaa acg cac ctg gcc tct ttt ctttca gcc ttc gcg 1392 Leu Gln Leu His Lys Thr His Leu Ala Ser Phe Leu SerAla Phe Ala 450 455 460 cgc caa gaa ctc tac ctc atg ggc agc ctc gtc cactcc atg ttg gta 1440 Arg Gln Glu Leu Tyr Leu Met Gly Ser Leu Val His SerMet Leu Val 465 470 475 480 cat acg acg gag aga cgc gaa atc ttc atc gtagaa acg ggc ctc tgt 1488 His Thr Thr Glu Arg Arg Glu Ile Phe Ile Val GluThr Gly Leu Cys 485 490 495 tca ttg gcc gag cta tca cac ttt acg cag ttgcta gct cat ccg cac 1536 Ser Leu Ala Glu Leu Ser His Phe Thr Gln Leu LeuAla His Pro His 500 505 510 cac gaa tac ctc agc gac ctg tac aca ccc tgttcc agt agc ggg cga 1584 His Glu Tyr Leu Ser Asp Leu Tyr Thr Pro Cys SerSer Ser Gly Arg 515 520 525 cgc gat cac tcg ctc gaa cgc ctc acg cgt ctcttc ccc gat gcc acc 1632 Arg Asp His Ser Leu Glu Arg Leu Thr Arg Leu PhePro Asp Ala Thr 530 535 540 gtt cct gct acc gtt ccc gcc gcc ctc tcc atccta tct acc atg caa 1680 Val Pro Ala Thr Val Pro Ala Ala Leu Ser Ile LeuSer Thr Met Gln 545 550 555 560 cca agc acg ctg gaa acc ttc ccc gac ctgttt tgt ctg ccg ctc ggc 1728 Pro Ser Thr Leu Glu Thr Phe Pro Asp Leu PheCys Leu Pro Leu Gly 565 570 575 gaa tcc ttc tcc gcg cta acc gtc tcc gaacac gtc agt tat gtc gta 1776 Glu Ser Phe Ser Ala Leu Thr Val Ser Glu HisVal Ser Tyr Val Val 580 585 590 aca aac cag tac ctg atc aaa ggt atc tcctac cct gtc tcc acc acc 1824 Thr Asn Gln Tyr Leu Ile Lys Gly Ile Ser TyrPro Val Ser Thr Thr 595 600 605 gtc gta ggc cag agc ctc atc atc acc caaacg gac agt caa act aaa 1872 Val Val Gly Gln Ser Leu Ile Ile Thr Gln ThrAsp Ser Gln Thr Lys 610 615 620 tgc gaa cta acg cgc aac atg cac acc acacac agc atc aca gcg gcg 1920 Cys Glu Leu Thr Arg Asn Met His Thr Thr HisSer Ile Thr Ala Ala 625 630 635 640 ctc aac att tca cta gaa aac tgc gccttt tgc caa agc gcc ctg cta 1968 Leu Asn Ile Ser Leu Glu Asn Cys Ala PheCys Gln Ser Ala Leu Leu 645 650 655 gaa tac gac gac acg caa ggc gtc atcaac atc atg tac atg cac gac 2016 Glu Tyr Asp Asp Thr Gln Gly Val Ile AsnIle Met Tyr Met His Asp 660 665 670 tcg gac gac gtc ctt ttc gcc ctg gatccc tac aac gaa gtg gtg gtc 2064 Ser Asp Asp Val Leu Phe Ala Leu Asp ProTyr Asn Glu Val Val Val 675 680 685 tca tct ccg cga act cac tac ctc atgctt ttg aaa aac ggt acg gtc 2112 Ser Ser Pro Arg Thr His Tyr Leu Met LeuLeu Lys Asn Gly Thr Val 690 695 700 cta gaa gta act gac gtc gtc gtg gacgcc acc gac agt cgt ctc ctc 2160 Leu Glu Val Thr Asp Val Val Val Asp AlaThr Asp Ser Arg Leu Leu 705 710 715 720 atg atg tcc gtc tac gcg cta tcggcc atc atc ggc atc tat ctg ctc 2208 Met Met Ser Val Tyr Ala Leu Ser AlaIle Ile Gly Ile Tyr Leu Leu 725 730 735 tac cgc atg ctc aag aca tgc tga2232 Tyr Arg Met Leu Lys Thr Cys 740 8 743 PRT Human cytomegalovirus 8Met Arg Pro Gly Leu Pro Pro Tyr Leu Thr Val Phe Thr Val Tyr Leu 1 5 1015 Leu Ser His Leu Pro Ser Gln Arg Tyr Gly Ala Asp Ala Ala Ser Glu 20 2530 Ala Leu Asp Pro His Ala Phe His Leu Leu Leu Asn Thr Tyr Gly Arg 35 4045 Pro Ile Arg Phe Leu Arg Glu Asn Thr Thr Gln Cys Thr Tyr Asn Ser 50 5560 Ser Leu Arg Asn Ser Thr Val Val Arg Glu Asn Ala Ile Ser Phe Asn 65 7075 80 Phe Phe Gln Ser Tyr Asn Gln Tyr Tyr Val Phe His Met Pro Arg Cys 8590 95 Leu Phe Ala Gly Pro Leu Ala Glu Gln Phe Leu Asn Gln Val Asp Leu100 105 110 Thr Glu Thr Leu Glu Arg Tyr Gln Gln Arg Leu Asn Thr Tyr AlaLeu 115 120 125 Val Ser Lys Asp Leu Ala Ser Tyr Arg Ser Phe Ser Gln GlnLeu Lys 130 135 140 Ala Gln Asp Ser Leu Gly Gln Gln Pro Thr Thr Val ProPro Pro Ile 145 150 155 160 Asp Leu Ser Ile Pro His Val Trp Met Pro ProGln Thr Thr Pro His 165 170 175 Asp Trp Lys Gly Ser His Thr Thr Ser GlyLeu His Arg Pro His Phe 180 185 190 Asn Gln Thr Cys Ile Leu Phe Asp GlyHis Asp Leu Leu Phe Ser Thr 195 200 205 Val Thr Pro Cys Leu His Gln GlyPhe Tyr Leu Met Asp Glu Leu Arg 210 215 220 Tyr Val Lys Ile Thr Leu ThrGlu Asp Phe Phe Val Val Thr Val Ser 225 230 235 240 Ile Asp Asp Asp ThrPro Met Leu Leu Ile Phe Gly His Leu Pro Arg 245 250 255 Val Leu Phe LysAla Pro Tyr Gln Arg Asp Asn Phe Ile Leu Arg Gln 260 265 270 Thr Glu LysHis Glu Leu Leu Val Leu Val Lys Lys Ala Gln Leu Asn 275 280 285 Arg HisSer Tyr Leu Lys Asp Ser Asp Phe Leu Asp Ala Ala Leu Asp 290 295 300 PheAsn Tyr Leu Asp Leu Ser Ala Leu Leu Arg Asn Ser Phe His Arg 305 310 315320 Tyr Ala Val Asp Val Leu Lys Ser Gly Arg Cys Gln Met Leu Asp Arg 325330 335 Arg Thr Val Glu Met Ala Phe Ala Tyr Ala Leu Ala Leu Phe Ala Ala340 345 350 Ala Arg Gln Glu Glu Ala Gly Thr Glu Ile Ser Ile Pro Arg AlaLeu 355 360 365 Asp Arg Gln Ala Ala Leu Leu Gln Ile Gln Glu Phe Met IleThr Cys 370 375 380 Leu Ser Gln Thr Pro Pro Arg Thr Thr Leu Leu Leu TyrPro Thr Ala 385 390 395 400 Val Asp Leu Ala Lys Arg Ala Leu Trp Thr ProAsp Gln Ile Thr Asp 405 410 415 Ile Thr Ser Leu Val Arg Leu Val Tyr IleLeu Ser Lys Gln Asn Gln 420 425 430 Gln His Leu Ile Pro Gln Trp Ala LeuArg Gln Ile Ala Asp Phe Ala 435 440 445 Leu Gln Leu His Lys Thr His LeuAla Ser Phe Leu Ser Ala Phe Ala 450 455 460 Arg Gln Glu Leu Tyr Leu MetGly Ser Leu Val His Ser Met Leu Val 465 470 475 480 His Thr Thr Glu ArgArg Glu Ile Phe Ile Val Glu Thr Gly Leu Cys 485 490 495 Ser Leu Ala GluLeu Ser His Phe Thr Gln Leu Leu Ala His Pro His 500 505 510 His Glu TyrLeu Ser Asp Leu Tyr Thr Pro Cys Ser Ser Ser Gly Arg 515 520 525 Arg AspHis Ser Leu Glu Arg Leu Thr Arg Leu Phe Pro Asp Ala Thr 530 535 540 ValPro Ala Thr Val Pro Ala Ala Leu Ser Ile Leu Ser Thr Met Gln 545 550 555560 Pro Ser Thr Leu Glu Thr Phe Pro Asp Leu Phe Cys Leu Pro Leu Gly 565570 575 Glu Ser Phe Ser Ala Leu Thr Val Ser Glu His Val Ser Tyr Val Val580 585 590 Thr Asn Gln Tyr Leu Ile Lys Gly Ile Ser Tyr Pro Val Ser ThrThr 595 600 605 Val Val Gly Gln Ser Leu Ile Ile Thr Gln Thr Asp Ser GlnThr Lys 610 615 620 Cys Glu Leu Thr Arg Asn Met His Thr Thr His Ser IleThr Ala Ala 625 630 635 640 Leu Asn Ile Ser Leu Glu Asn Cys Ala Phe CysGln Ser Ala Leu Leu 645 650 655 Glu Tyr Asp Asp Thr Gln Gly Val Ile AsnIle Met Tyr Met His Asp 660 665 670 Ser Asp Asp Val Leu Phe Ala Leu AspPro Tyr Asn Glu Val Val Val 675 680 685 Ser Ser Pro Arg Thr His Tyr LeuMet Leu Leu Lys Asn Gly Thr Val 690 695 700 Leu Glu Val Thr Asp Val ValVal Asp Ala Thr Asp Ser Arg Leu Leu 705 710 715 720 Met Met Ser Val TyrAla Leu Ser Ala Ile Ile Gly Ile Tyr Leu Leu 725 730 735 Tyr Arg Met LeuLys Thr Cys 740 9 921 DNA Human cytomegalovirus CDS (1)...(918) 9 cgtttt agg gat cga aga cct gag cgc caa ctt tcg gcg cca act ggc 48 Arg PheArg Asp Arg Arg Pro Glu Arg Gln Leu Ser Ala Pro Thr Gly 1 5 10 15 tcctta ccg tca cac tct cat cgt gcc gca gac ttg atg tgc cgc cgc 96 Ser LeuPro Ser His Ser His Arg Ala Ala Asp Leu Met Cys Arg Arg 20 25 30 ccg gattgc ggc ttc tct ttc tca cct gga ccg gtg gta ctg ctg tgg 144 Pro Asp CysGly Phe Ser Phe Ser Pro Gly Pro Val Val Leu Leu Trp 35 40 45 tgt tgc cttctg ctg ccc att gtt tcc tca gtc gcc gtc agc gtc gct 192 Cys Cys Leu LeuLeu Pro Ile Val Ser Ser Val Ala Val Ser Val Ala 50 55 60 cct acc gcc gccgag aaa gtc ccc gcg gag tgc ccc gaa cta acg cgt 240 Pro Thr Ala Ala GluLys Val Pro Ala Glu Cys Pro Glu Leu Thr Arg 65 70 75 80 cga tgc ctg ttgggt gag gtg ttt cag ggt gac aag tat gaa agt tgg 288 Arg Cys Leu Leu GlyGlu Val Phe Gln Gly Asp Lys Tyr Glu Ser Trp 85 90 95 ctg cgc ccg ttg gtgaat gtt acc aga cgc gat ggc ccg cta tcg caa 336 Leu Arg Pro Leu Val AsnVal Thr Arg Arg Asp Gly Pro Leu Ser Gln 100 105 110 ctt att cgt tac cgtccc gtt acg ccg gag gcc gcc aac tcc gtg ctg 384 Leu Ile Arg Tyr Arg ProVal Thr Pro Glu Ala Ala Asn Ser Val Leu 115 120 125 ttg gac gat gct ttcctg gac act ctg gcc ctg ctg tac aac aat ccg 432 Leu Asp Asp Ala Phe LeuAsp Thr Leu Ala Leu Leu Tyr Asn Asn Pro 130 135 140 gat caa ttg cgg gccctg ctg acg ctg ttg agc tcg gac aca gcg ccg 480 Asp Gln Leu Arg Ala LeuLeu Thr Leu Leu Ser Ser Asp Thr Ala Pro 145 150 155 160 cgc tgg atg acggtg atg cgc ggc tac agc gag tgc ggc gat ggc tcg 528 Arg Trp Met Thr ValMet Arg Gly Tyr Ser Glu Cys Gly Asp Gly Ser 165 170 175 ccg gcc gtg tacacg tgc gtg gac gac ctg tgc cgc ggc tac gac ctc 576 Pro Ala Val Tyr ThrCys Val Asp Asp Leu Cys Arg Gly Tyr Asp Leu 180 185 190 acg cga ctg tcatac ggg cgc agc atc ttc acg gaa cac gtg tta ggc 624 Thr Arg Leu Ser TyrGly Arg Ser Ile Phe Thr Glu His Val Leu Gly 195 200 205 ttc gag ctg gtgcca ccg tct ctc ttt aac gtg gtg gtg gcc ata cgc 672 Phe Glu Leu Val ProPro Ser Leu Phe Asn Val Val Val Ala Ile Arg 210 215 220 aac gaa gcc acgcgt acc aac cgc gcc gtg cgt ctg ccc gtg agc acc 720 Asn Glu Ala Thr ArgThr Asn Arg Ala Val Arg Leu Pro Val Ser Thr 225 230 235 240 gct gcc gcgccc gag ggc atc aca ctc ttt tac ggc ctg tac aac gca 768 Ala Ala Ala ProGlu Gly Ile Thr Leu Phe Tyr Gly Leu Tyr Asn Ala 245 250 255 gtg aag gaattc tgc ctg cgt cac cag ctg gac ccg ccg cta cta cgc 816 Val Lys Glu PheCys Leu Arg His Gln Leu Asp Pro Pro Leu Leu Arg 260 265 270 cac cta gataaa tac tac gcc gga ctg ccg ccc gag ctg aag cag acg 864 His Leu Asp LysTyr Tyr Ala Gly Leu Pro Pro Glu Leu Lys Gln Thr 275 280 285 cgc gtc aacctg ccg gct cac tcg cgc tat ggc cct caa gca gtg gat 912 Arg Val Asn LeuPro Ala His Ser Arg Tyr Gly Pro Gln Ala Val Asp 290 295 300 gct cgc taa921 Ala Arg 305 10 306 PRT Human cytomegalovirus 10 Arg Phe Arg Asp ArgArg Pro Glu Arg Gln Leu Ser Ala Pro Thr Gly 1 5 10 15 Ser Leu Pro SerHis Ser His Arg Ala Ala Asp Leu Met Cys Arg Arg 20 25 30 Pro Asp Cys GlyPhe Ser Phe Ser Pro Gly Pro Val Val Leu Leu Trp 35 40 45 Cys Cys Leu LeuLeu Pro Ile Val Ser Ser Val Ala Val Ser Val Ala 50 55 60 Pro Thr Ala AlaGlu Lys Val Pro Ala Glu Cys Pro Glu Leu Thr Arg 65 70 75 80 Arg Cys LeuLeu Gly Glu Val Phe Gln Gly Asp Lys Tyr Glu Ser Trp 85 90 95 Leu Arg ProLeu Val Asn Val Thr Arg Arg Asp Gly Pro Leu Ser Gln 100 105 110 Leu IleArg Tyr Arg Pro Val Thr Pro Glu Ala Ala Asn Ser Val Leu 115 120 125 LeuAsp Asp Ala Phe Leu Asp Thr Leu Ala Leu Leu Tyr Asn Asn Pro 130 135 140Asp Gln Leu Arg Ala Leu Leu Thr Leu Leu Ser Ser Asp Thr Ala Pro 145 150155 160 Arg Trp Met Thr Val Met Arg Gly Tyr Ser Glu Cys Gly Asp Gly Ser165 170 175 Pro Ala Val Tyr Thr Cys Val Asp Asp Leu Cys Arg Gly Tyr AspLeu 180 185 190 Thr Arg Leu Ser Tyr Gly Arg Ser Ile Phe Thr Glu His ValLeu Gly 195 200 205 Phe Glu Leu Val Pro Pro Ser Leu Phe Asn Val Val ValAla Ile Arg 210 215 220 Asn Glu Ala Thr Arg Thr Asn Arg Ala Val Arg LeuPro Val Ser Thr 225 230 235 240 Ala Ala Ala Pro Glu Gly Ile Thr Leu PheTyr Gly Leu Tyr Asn Ala 245 250 255 Val Lys Glu Phe Cys Leu Arg His GlnLeu Asp Pro Pro Leu Leu Arg 260 265 270 His Leu Asp Lys Tyr Tyr Ala GlyLeu Pro Pro Glu Leu Lys Gln Thr 275 280 285 Arg Val Asn Leu Pro Ala HisSer Arg Tyr Gly Pro Gln Ala Val Asp 290 295 300 Ala Arg 305 11 1401 DNAHuman cytomegalovirus CDS (1)...(1398) 11 atg ggg aga aaa gag atg atggtg aga gac gtc cct aag atg gtg ttt 48 Met Gly Arg Lys Glu Met Met ValArg Asp Val Pro Lys Met Val Phe 1 5 10 15 cta ata tct ata tct ttc ttgctt gtt tct ttc ata aac tgt aaa gtt 96 Leu Ile Ser Ile Ser Phe Leu LeuVal Ser Phe Ile Asn Cys Lys Val 20 25 30 atg tca aaa gcg ctt tat aat cgtcct tgg agg ggc ttg gta ctg tct 144 Met Ser Lys Ala Leu Tyr Asn Arg ProTrp Arg Gly Leu Val Leu Ser 35 40 45 aag ata ggc aaa tat aaa tta gat cagctt aag tta gaa att ttg aga 192 Lys Ile Gly Lys Tyr Lys Leu Asp Gln LeuLys Leu Glu Ile Leu Arg 50 55 60 caa cta gaa acg act att tct aca aaa tacaat gta agt aaa caa ccg 240 Gln Leu Glu Thr Thr Ile Ser Thr Lys Tyr AsnVal Ser Lys Gln Pro 65 70 75 80 gtt aaa aat ctc act atg aac atg aca gagttt cca caa tac tac att 288 Val Lys Asn Leu Thr Met Asn Met Thr Glu PhePro Gln Tyr Tyr Ile 85 90 95 tta gcg ggc ccc att cag aat tat agt ata acctat ctg tgg ttt gat 336 Leu Ala Gly Pro Ile Gln Asn Tyr Ser Ile Thr TyrLeu Trp Phe Asp 100 105 110 ttt tat agt acc cag ctt aga aaa ccc gca aaatac gtt tac tca cag 384 Phe Tyr Ser Thr Gln Leu Arg Lys Pro Ala Lys TyrVal Tyr Ser Gln 115 120 125 tac aat cat acg gct aaa acg ata aca ttc agaccc cca cct tgt ggt 432 Tyr Asn His Thr Ala Lys Thr Ile Thr Phe Arg ProPro Pro Cys Gly 130 135 140 act gtg cct tcc atg act tgt ctt tcc gaa atgcta aac gtt tcc aaa 480 Thr Val Pro Ser Met Thr Cys Leu Ser Glu Met LeuAsn Val Ser Lys 145 150 155 160 cgt aat gat act ggc gaa caa ggt tgc ggtaat ttc acc acg ttc aac 528 Arg Asn Asp Thr Gly Glu Gln Gly Cys Gly AsnPhe Thr Thr Phe Asn 165 170 175 ccc atg ttt ttc aat gta ccg cgt tgg aacacc aaa ttg tac gtg ggt 576 Pro Met Phe Phe Asn Val Pro Arg Trp Asn ThrLys Leu Tyr Val Gly 180 185 190 ccg act aag gtt aac gta gat agt caa acgatt tat ttt cta ggt tta 624 Pro Thr Lys Val Asn Val Asp Ser Gln Thr IleTyr Phe Leu Gly Leu 195 200 205 acc gcc ctg ctt tta cgt tac gca caa cgcaac tgt aca cac agt ttc 672 Thr Ala Leu Leu Leu Arg Tyr Ala Gln Arg AsnCys Thr His Ser Phe 210 215 220 tac ctg gtt aac gcc atg agc cgg aat ctattt cgc gtc ccc aag tat 720 Tyr Leu Val Asn Ala Met Ser Arg Asn Leu PheArg Val Pro Lys Tyr 225 230 235 240 att aac ggc acc aag tta aaa aac actatg cga aaa cta aaa cgt aaa 768 Ile Asn Gly Thr Lys Leu Lys Asn Thr MetArg Lys Leu Lys Arg Lys 245 250 255 caa gcg ccc gtt aag gaa caa ttc gaaaaa aaa gct aag aaa act cag 816 Gln Ala Pro Val Lys Glu Gln Phe Glu LysLys Ala Lys Lys Thr Gln 260 265 270 agt act act acg cca tac ttt tcc tataca acg tct gcc gct ctc aac 864 Ser Thr Thr Thr Pro Tyr Phe Ser Tyr ThrThr Ser Ala Ala Leu Asn 275 280 285 gtc act act aac gtg act tat agt attact acc gcc gca agg cgg gtt 912 Val Thr Thr Asn Val Thr Tyr Ser Ile ThrThr Ala Ala Arg Arg Val 290 295 300 tcc acg tct aca att gct tat cgt cctgat agc agc ttt atg aag tcc 960 Ser Thr Ser Thr Ile Ala Tyr Arg Pro AspSer Ser Phe Met Lys Ser 305 310 315 320 att atg gcc aca cag tta agg gaccta gca acg tgg gtg tat acc act 1008 Ile Met Ala Thr Gln Leu Arg Asp LeuAla Thr Trp Val Tyr Thr Thr 325 330 335 cta cgt tac cgg caa aat cct ttttgt gaa cca agc cgc aac cga acc 1056 Leu Arg Tyr Arg Gln Asn Pro Phe CysGlu Pro Ser Arg Asn Arg Thr 340 345 350 gcc gtg tca gaa ttt atg aaa aacacg cac gta cta atc cgt aac gaa 1104 Ala Val Ser Glu Phe Met Lys Asn ThrHis Val Leu Ile Arg Asn Glu 355 360 365 acg ccg tac act att tac ggt actctc gac atg agc tcc tta tat tac 1152 Thr Pro Tyr Thr Ile Tyr Gly Thr LeuAsp Met Ser Ser Leu Tyr Tyr 370 375 380 aac gaa acc atg ttc gtg gaa aacaaa aca gct tcc gat agt aac aaa 1200 Asn Glu Thr Met Phe Val Glu Asn LysThr Ala Ser Asp Ser Asn Lys 385 390 395 400 act aca cct acg tca cca tcaatg ggg ttt cag aga aca ttt ata gat 1248 Thr Thr Pro Thr Ser Pro Ser MetGly Phe Gln Arg Thr Phe Ile Asp 405 410 415 ccc ctg tgg gac tat cta gactcg ctg ctg ttt cta gat gag att cgt 1296 Pro Leu Trp Asp Tyr Leu Asp SerLeu Leu Phe Leu Asp Glu Ile Arg 420 425 430 aac ttt agc ctc cgg tca cccacg tat gta aac ctt acc ccg ccg gaa 1344 Asn Phe Ser Leu Arg Ser Pro ThrTyr Val Asn Leu Thr Pro Pro Glu 435 440 445 cac cgc cgg gct gta aat ctgtcc acc ctc aat agc ctt tgg tgg tgg 1392 His Arg Arg Ala Val Asn Leu SerThr Leu Asn Ser Leu Trp Trp Trp 450 455 460 ttg cag taa 1401 Leu Gln 46512 466 PRT Human cytomegalovirus 12 Met Gly Arg Lys Glu Met Met Val ArgAsp Val Pro Lys Met Val Phe 1 5 10 15 Leu Ile Ser Ile Ser Phe Leu LeuVal Ser Phe Ile Asn Cys Lys Val 20 25 30 Met Ser Lys Ala Leu Tyr Asn ArgPro Trp Arg Gly Leu Val Leu Ser 35 40 45 Lys Ile Gly Lys Tyr Lys Leu AspGln Leu Lys Leu Glu Ile Leu Arg 50 55 60 Gln Leu Glu Thr Thr Ile Ser ThrLys Tyr Asn Val Ser Lys Gln Pro 65 70 75 80 Val Lys Asn Leu Thr Met AsnMet Thr Glu Phe Pro Gln Tyr Tyr Ile 85 90 95 Leu Ala Gly Pro Ile Gln AsnTyr Ser Ile Thr Tyr Leu Trp Phe Asp 100 105 110 Phe Tyr Ser Thr Gln LeuArg Lys Pro Ala Lys Tyr Val Tyr Ser Gln 115 120 125 Tyr Asn His Thr AlaLys Thr Ile Thr Phe Arg Pro Pro Pro Cys Gly 130 135 140 Thr Val Pro SerMet Thr Cys Leu Ser Glu Met Leu Asn Val Ser Lys 145 150 155 160 Arg AsnAsp Thr Gly Glu Gln Gly Cys Gly Asn Phe Thr Thr Phe Asn 165 170 175 ProMet Phe Phe Asn Val Pro Arg Trp Asn Thr Lys Leu Tyr Val Gly 180 185 190Pro Thr Lys Val Asn Val Asp Ser Gln Thr Ile Tyr Phe Leu Gly Leu 195 200205 Thr Ala Leu Leu Leu Arg Tyr Ala Gln Arg Asn Cys Thr His Ser Phe 210215 220 Tyr Leu Val Asn Ala Met Ser Arg Asn Leu Phe Arg Val Pro Lys Tyr225 230 235 240 Ile Asn Gly Thr Lys Leu Lys Asn Thr Met Arg Lys Leu LysArg Lys 245 250 255 Gln Ala Pro Val Lys Glu Gln Phe Glu Lys Lys Ala LysLys Thr Gln 260 265 270 Ser Thr Thr Thr Pro Tyr Phe Ser Tyr Thr Thr SerAla Ala Leu Asn 275 280 285 Val Thr Thr Asn Val Thr Tyr Ser Ile Thr ThrAla Ala Arg Arg Val 290 295 300 Ser Thr Ser Thr Ile Ala Tyr Arg Pro AspSer Ser Phe Met Lys Ser 305 310 315 320 Ile Met Ala Thr Gln Leu Arg AspLeu Ala Thr Trp Val Tyr Thr Thr 325 330 335 Leu Arg Tyr Arg Gln Asn ProPhe Cys Glu Pro Ser Arg Asn Arg Thr 340 345 350 Ala Val Ser Glu Phe MetLys Asn Thr His Val Leu Ile Arg Asn Glu 355 360 365 Thr Pro Tyr Thr IleTyr Gly Thr Leu Asp Met Ser Ser Leu Tyr Tyr 370 375 380 Asn Glu Thr MetPhe Val Glu Asn Lys Thr Ala Ser Asp Ser Asn Lys 385 390 395 400 Thr ThrPro Thr Ser Pro Ser Met Gly Phe Gln Arg Thr Phe Ile Asp 405 410 415 ProLeu Trp Asp Tyr Leu Asp Ser Leu Leu Phe Leu Asp Glu Ile Arg 420 425 430Asn Phe Ser Leu Arg Ser Pro Thr Tyr Val Asn Leu Thr Pro Pro Glu 435 440445 His Arg Arg Ala Val Asn Leu Ser Thr Leu Asn Ser Leu Trp Trp Trp 450455 460 Leu Gln 465 13 1686 DNA Human cytomegalovirus CDS (1)...(1683)13 atg gag tcg cgc ggt cgc cgt tgt ccc gaa atg ata tcc gta ctg ggt 48Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly 1 5 1015 ccc att tcg ggg cac gtg ctg aaa gcc gtg ttt agt cgc ggc gat acg 96Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr 20 25 30ccg gtg ctg ccg cac gag acg cga ctc ctg cag acg ggt atc cac gta 144 ProVal Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val 35 40 45 cgcgtg agc cag ccc tcg ctg atc ttg gta tcg cag tac acg ccc gac 192 Arg ValSer Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp 50 55 60 tcg acgcca tgc cac cgc ggc gac aat cag ctg cag gtg cag cac acg 240 Ser Thr ProCys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr 65 70 75 80 tac tttacg ggc agc gag gtg gag aac gtg tcg gtc aac gtg cac aac 288 Tyr Phe ThrGly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn 85 90 95 ccc acg ggccga agc atc tgc ccc agc cag gag ccc atg tcg atc tat 336 Pro Thr Gly ArgSer Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr 100 105 110 gtg tac gcgctg ccg ctc aag atg ctg aac atc ccc agc atc aac gtg 384 Val Tyr Ala LeuPro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val 115 120 125 cac cac tacccg tcg gcg gcc gag cgc aaa cac cga cac ctg ccc gta 432 His His Tyr ProSer Ala Ala Glu Arg Lys His Arg His Leu Pro Val 130 135 140 gct gac gctgtg att cac gcg tcg ggc aag cag atg tgg cag gcg cgt 480 Ala Asp Ala ValIle His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg 145 150 155 160 ctc acggtc tcg gga ctg gcc tgg acg cgt cag cag aac cag tgg aaa 528 Leu Thr ValSer Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys 165 170 175 gag cccgac gtc tac tac acg tca gcg ttc gtg ttt ccc acc aag gac 576 Glu Pro AspVal Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp 180 185 190 gtg gcactg cgg cac gtg gtg tgc gcg cac gag ctg gtt tgc tcc atg 624 Val Ala LeuArg His Val Val Cys Ala His Glu Leu Val Cys Ser Met 195 200 205 gag aacacg cgc gca acc aag atg cag gtg ata ggt gac cag tac gtc 672 Glu Asn ThrArg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val 210 215 220 aag gtgtac ctg gag tcc ttc tgc gag gac gtg ccc tcc ggc aag ctc 720 Lys Val TyrLeu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu 225 230 235 240 tttatg cac gtc acg ctg ggc tct gac gtg gaa gag gac ctg acg atg 768 Phe MetHis Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met 245 250 255 acccgc aac ccg caa ccc ttc atg cgc ccc cac gag cgc aac ggc ttt 816 Thr ArgAsn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe 260 265 270 acggtg ttg tgt ccc aaa aat atg ata atc aaa ccg ggc aag atc tcg 864 Thr ValLeu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser 275 280 285 cacatc atg ctg gat gtg gct ttt acc tca cac gag cat ttt ggg ctg 912 His IleMet Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu 290 295 300 ctgtgt ccc aag agc atc ccg ggc ctg agc atc tca ggt aac ctg ttg 960 Leu CysPro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu 305 310 315 320atg aac ggg cag cag atc ttc ctg gag gta caa gcc ata cgc gag acc 1008 MetAsn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr 325 330 335gtg gaa ctg cgt cag tac gat ccc gtg gct gcg ctc ttc ttt ttc gat 1056 ValGlu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp 340 345 350atc gac ttg ctg ctg cag cgc ggg cct cag tac agc gag cac ccc acc 1104 IleAsp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr 355 360 365ttc acc agc cag tat cgc atc cag ggc aag ctt gag tac cga cac acc 1152 PheThr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr 370 375 380tgg gac cgg cac gac gag ggt gcc gcc cag ggc gac gac gac gtc tgg 1200 TrpAsp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp 385 390 395400 acc agc gga tcg gac tcc gac gaa gaa ctc gta acc acc gag cgc aag 1248Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys 405 410415 acg ccc cgc gtc acc ggc ggc ggc gcc atg gcg ggc gcc tcc act tcc 1296Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr Ser 420 425430 gcg ggc cgc aaa cgc aaa tca gca tcc tcg gcg acg gcg tgc acg tcg 1344Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ser 435 440445 ggc gtt atg aca cgc ggc cgc ctt aag gcc gag tcc acc gtc gcg ccc 1392Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro 450 455460 gaa gag gac acc gac gag gat tcc gac aac gaa atc cac aat ccg gcc 1440Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala 465 470475 480 gtg ttc acc tgg ccg ccc tgg cag gcc ggc atc ctg gcc cgc aac ctg1488 Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu 485490 495 gtg ccc atg gtg gct acg gtt cag ggt cag aat ctg aag tac cag gaa1536 Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr Gln Glu 500505 510 ttc ttc tgg gac gcc aac gac atc tac cgc atc ttc gcc gaa ttg gaa1584 Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu 515520 525 ggc gta tgg cag ccc gct gcg caa ccc aaa cgt cgc cgc cac cgg caa1632 Gly Val Trp Gln Pro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln 530535 540 gac gcc ttg ccc ggg cca tgc atc gcc tcg acg ccc aaa aag cac cga1680 Asp Ala Leu Pro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His Arg 545550 555 560 ggt tga 1686 Gly 14 561 PRT Human cytomegalovirus 14 Met GluSer Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly 1 5 10 15 ProIle Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr 20 25 30 ProVal Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val 35 40 45 ArgVal Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp 50 55 60 SerThr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr 65 70 75 80Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn 85 90 95Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr 100 105110 Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val 115120 125 His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val130 135 140 Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln AlaArg 145 150 155 160 Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln AsnGln Trp Lys 165 170 175 Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val PhePro Thr Lys Asp 180 185 190 Val Ala Leu Arg His Val Val Cys Ala His GluLeu Val Cys Ser Met 195 200 205 Glu Asn Thr Arg Ala Thr Lys Met Gln ValIle Gly Asp Gln Tyr Val 210 215 220 Lys Val Tyr Leu Glu Ser Phe Cys GluAsp Val Pro Ser Gly Lys Leu 225 230 235 240 Phe Met His Val Thr Leu GlySer Asp Val Glu Glu Asp Leu Thr Met 245 250 255 Thr Arg Asn Pro Gln ProPhe Met Arg Pro His Glu Arg Asn Gly Phe 260 265 270 Thr Val Leu Cys ProLys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser 275 280 285 His Ile Met LeuAsp Val Ala Phe Thr Ser His Glu His Phe Gly Leu 290 295 300 Leu Cys ProLys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu 305 310 315 320 MetAsn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr 325 330 335Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp 340 345350 Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr 355360 365 Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr370 375 380 Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp ValTrp 385 390 395 400 Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr ThrGlu Arg Lys 405 410 415 Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala GlyAla Ser Thr Ser 420 425 430 Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser AlaThr Ala Cys Thr Ser 435 440 445 Gly Val Met Thr Arg Gly Arg Leu Lys AlaGlu Ser Thr Val Ala Pro 450 455 460 Glu Glu Asp Thr Asp Glu Asp Ser AspAsn Glu Ile His Asn Pro Ala 465 470 475 480 Val Phe Thr Trp Pro Pro TrpGln Ala Gly Ile Leu Ala Arg Asn Leu 485 490 495 Val Pro Met Val Ala ThrVal Gln Gly Gln Asn Leu Lys Tyr Gln Glu 500 505 510 Phe Phe Trp Asp AlaAsn Asp Ile Tyr Arg Ile Phe Ala Glu Leu Glu 515 520 525 Gly Val Trp GlnPro Ala Ala Gln Pro Lys Arg Arg Arg His Arg Gln 530 535 540 Asp Ala LeuPro Gly Pro Cys Ile Ala Ser Thr Pro Lys Lys His Arg 545 550 555 560 Gly15 3147 DNA Human cytomegalovirus CDS (1)...(3144) 15 atg agt ttg cagttt atc ggt cta cag cgg cgc gat gtg gta gcc ctg 48 Met Ser Leu Gln PheIle Gly Leu Gln Arg Arg Asp Val Val Ala Leu 1 5 10 15 gtc aac ttt ctgcgc cat ctc acg caa aag ccc gac gtg gat ctc gag 96 Val Asn Phe Leu ArgHis Leu Thr Gln Lys Pro Asp Val Asp Leu Glu 20 25 30 gca cac ccc aag atcctg aaa aaa tgt ggc gaa aaa cgc ctg cac cgg 144 Ala His Pro Lys Ile LeuLys Lys Cys Gly Glu Lys Arg Leu His Arg 35 40 45 cgt acg gtg ctg ttc aacgag ctc atg ctt tgg ttg gga tac tac cgc 192 Arg Thr Val Leu Phe Asn GluLeu Met Leu Trp Leu Gly Tyr Tyr Arg 50 55 60 gag ctg cgt ttt cac aac cccgac ctc tcc tca gtg ctc gag gag ttc 240 Glu Leu Arg Phe His Asn Pro AspLeu Ser Ser Val Leu Glu Glu Phe 65 70 75 80 gag gtg cgt tgc gtg gcc gtggcg cgt cgc ggc tac act tac ccg ttc 288 Glu Val Arg Cys Val Ala Val AlaArg Arg Gly Tyr Thr Tyr Pro Phe 85 90 95 ggt gat cgt ggt aag gcg cgt gaccac ctg gct gtg cta gac cgt acc 336 Gly Asp Arg Gly Lys Ala Arg Asp HisLeu Ala Val Leu Asp Arg Thr 100 105 110 gaa ttc gat acg gac gtg cgc cacgat gcc gag atc gtg gaa cgc gcg 384 Glu Phe Asp Thr Asp Val Arg His AspAla Glu Ile Val Glu Arg Ala 115 120 125 ctc gta agc gcg gtc att ctg gccaag atg tcg gtg cgc gag acg ctg 432 Leu Val Ser Ala Val Ile Leu Ala LysMet Ser Val Arg Glu Thr Leu 130 135 140 gtc aca gcc atc ggc cag acg gaaccc atc gcc ttt gtg cac ctc aag 480 Val Thr Ala Ile Gly Gln Thr Glu ProIle Ala Phe Val His Leu Lys 145 150 155 160 gat acg gag gtg cag cgc attgaa gaa aac ctg gag ggt gtg cgc cgt 528 Asp Thr Glu Val Gln Arg Ile GluGlu Asn Leu Glu Gly Val Arg Arg 165 170 175 aac atg ttc tgc gtg aaa ccgctc gac ctt aac ctg gac cgg cac gcc 576 Asn Met Phe Cys Val Lys Pro LeuAsp Leu Asn Leu Asp Arg His Ala 180 185 190 aac acg gcg ctg gtc aac gccgtc aac aag ctc gtg tac acg ggc cgt 624 Asn Thr Ala Leu Val Asn Ala ValAsn Lys Leu Val Tyr Thr Gly Arg 195 200 205 ctc atc atg aac gtg cgc aggtct tgg gag gag ctg gag cgc aaa tgt 672 Leu Ile Met Asn Val Arg Arg SerTrp Glu Glu Leu Glu Arg Lys Cys 210 215 220 ctg gcg cgc att cag gag cgctgc aag ctg ctg gtc aag gag ctg cgc 720 Leu Ala Arg Ile Gln Glu Arg CysLys Leu Leu Val Lys Glu Leu Arg 225 230 235 240 atg tgc ctt tcc ttt gattcc aac tac tgt cgc aat atc ctc aag cac 768 Met Cys Leu Ser Phe Asp SerAsn Tyr Cys Arg Asn Ile Leu Lys His 245 250 255 gcc gtg gaa aac ggc gactcg gcc gac acg ctg ttg gag ctg ctc atc 816 Ala Val Glu Asn Gly Asp SerAla Asp Thr Leu Leu Glu Leu Leu Ile 260 265 270 gag gac ttt gat atc tacgtg gac agc ttc cca cag tcg gcg cac acg 864 Glu Asp Phe Asp Ile Tyr ValAsp Ser Phe Pro Gln Ser Ala His Thr 275 280 285 ttt ttg ggc gcg cgc tcgccg tcg ttg gag ttt gac gat gac gcc aat 912 Phe Leu Gly Ala Arg Ser ProSer Leu Glu Phe Asp Asp Asp Ala Asn 290 295 300 ctc ctc tcg ctc ggc ggcggt tcg gcc ttc tcg tcg gta ccc aag aaa 960 Leu Leu Ser Leu Gly Gly GlySer Ala Phe Ser Ser Val Pro Lys Lys 305 310 315 320 cat gtc ccc acg cagccg ctg gac ggc tgg agc tgg atc gcc agt ccc 1008 His Val Pro Thr Gln ProLeu Asp Gly Trp Ser Trp Ile Ala Ser Pro 325 330 335 tgg aag gga cac aaaccg ttc cgc ttc gag gcc cat ggt tct ctg gca 1056 Trp Lys Gly His Lys ProPhe Arg Phe Glu Ala His Gly Ser Leu Ala 340 345 350 ccg gcc gcc gaa gcccac gct gcc cgt tcg gcg gcc gtc ggc tat tac 1104 Pro Ala Ala Glu Ala HisAla Ala Arg Ser Ala Ala Val Gly Tyr Tyr 355 360 365 gac gaa gag gaa aagcgt cgc gag cgg cag aaa cgg gtg gac gac gag 1152 Asp Glu Glu Glu Lys ArgArg Glu Arg Gln Lys Arg Val Asp Asp Glu 370 375 380 gtg gtg cag cgt gagaaa cag cag ctg aag gct tgg gag gag agg cag 1200 Val Val Gln Arg Glu LysGln Gln Leu Lys Ala Trp Glu Glu Arg Gln 385 390 395 400 cag aac ctg cagcaa cgt cag cag caa cca ccg ccc ccg gca cgt aaa 1248 Gln Asn Leu Gln GlnArg Gln Gln Gln Pro Pro Pro Pro Ala Arg Lys 405 410 415 ccg agc gcc tcccgg agg ctc ttt ggc tcc agt gcc gat gag gac gac 1296 Pro Ser Ala Ser ArgArg Leu Phe Gly Ser Ser Ala Asp Glu Asp Asp 420 425 430 gac gat gat gatgac gag aaa aac atc ttt acg ccc atc aag aaa ccg 1344 Asp Asp Asp Asp AspGlu Lys Asn Ile Phe Thr Pro Ile Lys Lys Pro 435 440 445 gga act agc ggcaag ggc gcc gct agt ggt ggc ggt gtt tcc agc att 1392 Gly Thr Ser Gly LysGly Ala Ala Ser Gly Gly Gly Val Ser Ser Ile 450 455 460 ttc agc ggc ctgtta tcc tcg ggc agt cag aaa ccg acc agc ggt ccc 1440 Phe Ser Gly Leu LeuSer Ser Gly Ser Gln Lys Pro Thr Ser Gly Pro 465 470 475 480 ttg aac atcccg caa caa caa cag cgt cac gcg gct ttc agt ctc gtc 1488 Leu Asn Ile ProGln Gln Gln Gln Arg His Ala Ala Phe Ser Leu Val 485 490 495 tcc ccg caggtg acc aag gcc agc ccg gga agg gtc cgt cgg gac agc 1536 Ser Pro Gln ValThr Lys Ala Ser Pro Gly Arg Val Arg Arg Asp Ser 500 505 510 gcg tgg gacgtg agg ccg ctc acg gag acc aga ggg gat ctt ttc tcg 1584 Ala Trp Asp ValArg Pro Leu Thr Glu Thr Arg Gly Asp Leu Phe Ser 515 520 525 ggc gac gaggat tcc gac agc tcg gat ggc tat ccc ccc aac cgt caa 1632 Gly Asp Glu AspSer Asp Ser Ser Asp Gly Tyr Pro Pro Asn Arg Gln 530 535 540 gat ccg cgtttc acc gac acg ctg gtg gac atc acg gat acc gag acg 1680 Asp Pro Arg PheThr Asp Thr Leu Val Asp Ile Thr Asp Thr Glu Thr 545 550 555 560 agc gccaaa ccg ccc gtc acc acc gcg tac aag ttc gag caa ccg acg 1728 Ser Ala LysPro Pro Val Thr Thr Ala Tyr Lys Phe Glu Gln Pro Thr 565 570 575 ttg acgttc ggc gcc gga gtt aac gtt cct gct ggc gcc ggc gct gcc 1776 Leu Thr PheGly Ala Gly Val Asn Val Pro Ala Gly Ala Gly Ala Ala 580 585 590 atc ctcacg ccg acg cct gtc aat cct tcc acg gcc ccc gct ccg gcc 1824 Ile Leu ThrPro Thr Pro Val Asn Pro Ser Thr Ala Pro Ala Pro Ala 595 600 605 ccg acacct acc ttc gcg ggt acc caa acc ccg gtc aac ggt aac tcg 1872 Pro Thr ProThr Phe Ala Gly Thr Gln Thr Pro Val Asn Gly Asn Ser 610 615 620 ccc tgggct ccg acg gcg ccg ttg ccc ggg gat atg aac ccc gcc aac 1920 Pro Trp AlaPro Thr Ala Pro Leu Pro Gly Asp Met Asn Pro Ala Asn 625 630 635 640 tggccg cgc gaa cgc gcg tgg gcc ctc aag aat cct cac ctg gct tac 1968 Trp ProArg Glu Arg Ala Trp Ala Leu Lys Asn Pro His Leu Ala Tyr 645 650 655 aatccc ttc agg atg cct acg act tcc acg gct tct caa aac acc gtg 2016 Asn ProPhe Arg Met Pro Thr Thr Ser Thr Ala Ser Gln Asn Thr Val 660 665 670 tccacc acc cct cgg agg ccg tcg act cca cgc gcc gcg gtg aca caa 2064 Ser ThrThr Pro Arg Arg Pro Ser Thr Pro Arg Ala Ala Val Thr Gln 675 680 685 acagcg tct cgg gac gcc gct gat gag gtt tgg gct tta agg gac caa 2112 Thr AlaSer Arg Asp Ala Ala Asp Glu Val Trp Ala Leu Arg Asp Gln 690 695 700 actgca gag tca ccg gtc gaa gac agc gag gag gaa gac gac gac tcc 2160 Thr AlaGlu Ser Pro Val Glu Asp Ser Glu Glu Glu Asp Asp Asp Ser 705 710 715 720tcg gac acc ggc tcc gtc gtc agc ctg gga cac aca aca ccg tcg tcc 2208 SerAsp Thr Gly Ser Val Val Ser Leu Gly His Thr Thr Pro Ser Ser 725 730 735gat tac aac aac gac gtc att tcg cct ccc agt cag acg ccc gag cag 2256 AspTyr Asn Asn Asp Val Ile Ser Pro Pro Ser Gln Thr Pro Glu Gln 740 745 750tcg acg ccg tcc aga ata cgt aaa gct aag tta tcg tct cca atg acg 2304 SerThr Pro Ser Arg Ile Arg Lys Ala Lys Leu Ser Ser Pro Met Thr 755 760 765acg aca tcc acg agc cag aaa ccg gtg ctg ggc aag cga gtc gcg acg 2352 ThrThr Ser Thr Ser Gln Lys Pro Val Leu Gly Lys Arg Val Ala Thr 770 775 780ccg cac gcg tcc gcc cga gcg cag acg gtg acg tcg acg ccg gtt cag 2400 ProHis Ala Ser Ala Arg Ala Gln Thr Val Thr Ser Thr Pro Val Gln 785 790 795800 gga agg cta gag aaa cag gtg tcg ggc acg ccg tcg acg gta ccc gcc 2448Gly Arg Leu Glu Lys Gln Val Ser Gly Thr Pro Ser Thr Val Pro Ala 805 810815 acg ctg ttg caa cct caa ccg gct tcg tct aaa acg acg tca tca agg 2496Thr Leu Leu Gln Pro Gln Pro Ala Ser Ser Lys Thr Thr Ser Ser Arg 820 825830 aac gtg act tct ggc gcg gga acc tct tcc gct tct tcg gct cga cag 2544Asn Val Thr Ser Gly Ala Gly Thr Ser Ser Ala Ser Ser Ala Arg Gln 835 840845 ccg tca gcc tcg gcg tcc gtt ttg tcg ccc acg gag gat gat gtc gtg 2592Pro Ser Ala Ser Ala Ser Val Leu Ser Pro Thr Glu Asp Asp Val Val 850 855860 tcc ccc gcc aca tcg ccg ctg tcc atg ctt tcg tca gcc tct ccg tcc 2640Ser Pro Ala Thr Ser Pro Leu Ser Met Leu Ser Ser Ala Ser Pro Ser 865 870875 880 ccg gcc aag agt gcc ccc ccg tct ccg gtg aaa ggc cgg ggc agc cgc2688 Pro Ala Lys Ser Ala Pro Pro Ser Pro Val Lys Gly Arg Gly Ser Arg 885890 895 gtc ggt gtt cct tcc ttg aaa cct act ttg ggc ggc aag gcg gtg gta2736 Val Gly Val Pro Ser Leu Lys Pro Thr Leu Gly Gly Lys Ala Val Val 900905 910 ggt cga ccg ccc tcg gtc ccc gtg agc ggt agc gcg ccg ggt cgc ctg2784 Gly Arg Pro Pro Ser Val Pro Val Ser Gly Ser Ala Pro Gly Arg Leu 915920 925 tcc ggc agc agc cgg gcc gcc tcg acc acg ccg acg tat ccc gcg gta2832 Ser Gly Ser Ser Arg Ala Ala Ser Thr Thr Pro Thr Tyr Pro Ala Val 930935 940 acc acc gtt tac cca ccg tcg tct acg gcc aaa agc agc gta tcg aat2880 Thr Thr Val Tyr Pro Pro Ser Ser Thr Ala Lys Ser Ser Val Ser Asn 945950 955 960 gcg ccg cct gtg gcc tcc ccc tcc atc ctg aaa ccg ggg gcg agcgcg 2928 Ala Pro Pro Val Ala Ser Pro Ser Ile Leu Lys Pro Gly Ala Ser Ala965 970 975 gct ttg caa tca cgc cgc tcg acg ggg acc gcc gcc gta ggt tccccc 2976 Ala Leu Gln Ser Arg Arg Ser Thr Gly Thr Ala Ala Val Gly Ser Pro980 985 990 gtc aag agc acg acg ggc atg aaa acg gtg gct ttc gac cta tcgtcg 3024 Val Lys Ser Thr Thr Gly Met Lys Thr Val Ala Phe Asp Leu Ser Ser995 1000 1005 ccc cag aag agc ggt acg ggg ccg caa ccg ggt tct gcc ggcatg ggg 3072 Pro Gln Lys Ser Gly Thr Gly Pro Gln Pro Gly Ser Ala Gly MetGly 1010 1015 1020 ggc gcc aaa acg ccg tcg gac gcc gtg cag aac atc ctccaa aag atc 3120 Gly Ala Lys Thr Pro Ser Asp Ala Val Gln Asn Ile Leu GlnLys Ile 1025 1030 1035 1040 gag aag att aag aac acg gag gaa tag 3147 GluLys Ile Lys Asn Thr Glu Glu 1045 16 1048 PRT Human cytomegalovirus 16Met Ser Leu Gln Phe Ile Gly Leu Gln Arg Arg Asp Val Val Ala Leu 1 5 1015 Val Asn Phe Leu Arg His Leu Thr Gln Lys Pro Asp Val Asp Leu Glu 20 2530 Ala His Pro Lys Ile Leu Lys Lys Cys Gly Glu Lys Arg Leu His Arg 35 4045 Arg Thr Val Leu Phe Asn Glu Leu Met Leu Trp Leu Gly Tyr Tyr Arg 50 5560 Glu Leu Arg Phe His Asn Pro Asp Leu Ser Ser Val Leu Glu Glu Phe 65 7075 80 Glu Val Arg Cys Val Ala Val Ala Arg Arg Gly Tyr Thr Tyr Pro Phe 8590 95 Gly Asp Arg Gly Lys Ala Arg Asp His Leu Ala Val Leu Asp Arg Thr100 105 110 Glu Phe Asp Thr Asp Val Arg His Asp Ala Glu Ile Val Glu ArgAla 115 120 125 Leu Val Ser Ala Val Ile Leu Ala Lys Met Ser Val Arg GluThr Leu 130 135 140 Val Thr Ala Ile Gly Gln Thr Glu Pro Ile Ala Phe ValHis Leu Lys 145 150 155 160 Asp Thr Glu Val Gln Arg Ile Glu Glu Asn LeuGlu Gly Val Arg Arg 165 170 175 Asn Met Phe Cys Val Lys Pro Leu Asp LeuAsn Leu Asp Arg His Ala 180 185 190 Asn Thr Ala Leu Val Asn Ala Val AsnLys Leu Val Tyr Thr Gly Arg 195 200 205 Leu Ile Met Asn Val Arg Arg SerTrp Glu Glu Leu Glu Arg Lys Cys 210 215 220 Leu Ala Arg Ile Gln Glu ArgCys Lys Leu Leu Val Lys Glu Leu Arg 225 230 235 240 Met Cys Leu Ser PheAsp Ser Asn Tyr Cys Arg Asn Ile Leu Lys His 245 250 255 Ala Val Glu AsnGly Asp Ser Ala Asp Thr Leu Leu Glu Leu Leu Ile 260 265 270 Glu Asp PheAsp Ile Tyr Val Asp Ser Phe Pro Gln Ser Ala His Thr 275 280 285 Phe LeuGly Ala Arg Ser Pro Ser Leu Glu Phe Asp Asp Asp Ala Asn 290 295 300 LeuLeu Ser Leu Gly Gly Gly Ser Ala Phe Ser Ser Val Pro Lys Lys 305 310 315320 His Val Pro Thr Gln Pro Leu Asp Gly Trp Ser Trp Ile Ala Ser Pro 325330 335 Trp Lys Gly His Lys Pro Phe Arg Phe Glu Ala His Gly Ser Leu Ala340 345 350 Pro Ala Ala Glu Ala His Ala Ala Arg Ser Ala Ala Val Gly TyrTyr 355 360 365 Asp Glu Glu Glu Lys Arg Arg Glu Arg Gln Lys Arg Val AspAsp Glu 370 375 380 Val Val Gln Arg Glu Lys Gln Gln Leu Lys Ala Trp GluGlu Arg Gln 385 390 395 400 Gln Asn Leu Gln Gln Arg Gln Gln Gln Pro ProPro Pro Ala Arg Lys 405 410 415 Pro Ser Ala Ser Arg Arg Leu Phe Gly SerSer Ala Asp Glu Asp Asp 420 425 430 Asp Asp Asp Asp Asp Glu Lys Asn IlePhe Thr Pro Ile Lys Lys Pro 435 440 445 Gly Thr Ser Gly Lys Gly Ala AlaSer Gly Gly Gly Val Ser Ser Ile 450 455 460 Phe Ser Gly Leu Leu Ser SerGly Ser Gln Lys Pro Thr Ser Gly Pro 465 470 475 480 Leu Asn Ile Pro GlnGln Gln Gln Arg His Ala Ala Phe Ser Leu Val 485 490 495 Ser Pro Gln ValThr Lys Ala Ser Pro Gly Arg Val Arg Arg Asp Ser 500 505 510 Ala Trp AspVal Arg Pro Leu Thr Glu Thr Arg Gly Asp Leu Phe Ser 515 520 525 Gly AspGlu Asp Ser Asp Ser Ser Asp Gly Tyr Pro Pro Asn Arg Gln 530 535 540 AspPro Arg Phe Thr Asp Thr Leu Val Asp Ile Thr Asp Thr Glu Thr 545 550 555560 Ser Ala Lys Pro Pro Val Thr Thr Ala Tyr Lys Phe Glu Gln Pro Thr 565570 575 Leu Thr Phe Gly Ala Gly Val Asn Val Pro Ala Gly Ala Gly Ala Ala580 585 590 Ile Leu Thr Pro Thr Pro Val Asn Pro Ser Thr Ala Pro Ala ProAla 595 600 605 Pro Thr Pro Thr Phe Ala Gly Thr Gln Thr Pro Val Asn GlyAsn Ser 610 615 620 Pro Trp Ala Pro Thr Ala Pro Leu Pro Gly Asp Met AsnPro Ala Asn 625 630 635 640 Trp Pro Arg Glu Arg Ala Trp Ala Leu Lys AsnPro His Leu Ala Tyr 645 650 655 Asn Pro Phe Arg Met Pro Thr Thr Ser ThrAla Ser Gln Asn Thr Val 660 665 670 Ser Thr Thr Pro Arg Arg Pro Ser ThrPro Arg Ala Ala Val Thr Gln 675 680 685 Thr Ala Ser Arg Asp Ala Ala AspGlu Val Trp Ala Leu Arg Asp Gln 690 695 700 Thr Ala Glu Ser Pro Val GluAsp Ser Glu Glu Glu Asp Asp Asp Ser 705 710 715 720 Ser Asp Thr Gly SerVal Val Ser Leu Gly His Thr Thr Pro Ser Ser 725 730 735 Asp Tyr Asn AsnAsp Val Ile Ser Pro Pro Ser Gln Thr Pro Glu Gln 740 745 750 Ser Thr ProSer Arg Ile Arg Lys Ala Lys Leu Ser Ser Pro Met Thr 755 760 765 Thr ThrSer Thr Ser Gln Lys Pro Val Leu Gly Lys Arg Val Ala Thr 770 775 780 ProHis Ala Ser Ala Arg Ala Gln Thr Val Thr Ser Thr Pro Val Gln 785 790 795800 Gly Arg Leu Glu Lys Gln Val Ser Gly Thr Pro Ser Thr Val Pro Ala 805810 815 Thr Leu Leu Gln Pro Gln Pro Ala Ser Ser Lys Thr Thr Ser Ser Arg820 825 830 Asn Val Thr Ser Gly Ala Gly Thr Ser Ser Ala Ser Ser Ala ArgGln 835 840 845 Pro Ser Ala Ser Ala Ser Val Leu Ser Pro Thr Glu Asp AspVal Val 850 855 860 Ser Pro Ala Thr Ser Pro Leu Ser Met Leu Ser Ser AlaSer Pro Ser 865 870 875 880 Pro Ala Lys Ser Ala Pro Pro Ser Pro Val LysGly Arg Gly Ser Arg 885 890 895 Val Gly Val Pro Ser Leu Lys Pro Thr LeuGly Gly Lys Ala Val Val 900 905 910 Gly Arg Pro Pro Ser Val Pro Val SerGly Ser Ala Pro Gly Arg Leu 915 920 925 Ser Gly Ser Ser Arg Ala Ala SerThr Thr Pro Thr Tyr Pro Ala Val 930 935 940 Thr Thr Val Tyr Pro Pro SerSer Thr Ala Lys Ser Ser Val Ser Asn 945 950 955 960 Ala Pro Pro Val AlaSer Pro Ser Ile Leu Lys Pro Gly Ala Ser Ala 965 970 975 Ala Leu Gln SerArg Arg Ser Thr Gly Thr Ala Ala Val Gly Ser Pro 980 985 990 Val Lys SerThr Thr Gly Met Lys Thr Val Ala Phe Asp Leu Ser Ser 995 1000 1005 ProGln Lys Ser Gly Thr Gly Pro Gln Pro Gly Ser Ala Gly Met Gly 1010 10151020 Gly Ala Lys Thr Pro Ser Asp Ala Val Gln Asn Ile Leu Gln Lys Ile1025 1030 1035 1040 Glu Lys Ile Lys Asn Thr Glu Glu 1045

What is claimed is:
 1. A composition comprising a plurality of sets ofnucleic acid molecules, each set of nucleic acid molecules encoding adifferent type of cytomegalovirus (CMV) polypeptide, and each moleculeof a set encoding the same type of CMV polypeptide, wherein one or moresets of the plurality encodes a CMV polypeptide that induces aneutralizing antibody response, and one or more sets of the pluralityencodes a CMV polypeptide that induces a cell-mediated immune response.2. The composition of claim 1, wherein the nucleic acid moleculescomprise DNA plasmids.
 3. The composition of claim 1, wherein the CMVpolypeptides are human CMV (HCMV) polypeptides.
 4. The composition ofclaim 1, wherein the CMV polypeptides that induce an antibody responseare selected from the group consisting of glycoprotein B, glycoproteincomplex II, and glycoprotein complex III, and antigenic fragmentsthereof.
 5. The composition of claim 1, wherein the CMV polypeptidesthat induce an antibody response comprise glycoprotein B andglycoprotein complex II, or antigenic fragments thereof.
 6. Thecomposition of claim 1, wherein the CMV polypeptides that induce acell-mediated immune response are selected from the group consisting ofphosphoprotein pp65 (pp65), phosphoprotein pp150 (pp150), and antigenicfragments thereof.
 7. A composition comprising a plurality of sets ofnucleic acid molecules, each set encoding a different type of humancytomegalovirus (HCMV) polypeptide that induces a neutralizing antibodyresponse, and each nucleic acid molecule of a set encoding the same typeof HCMV polypeptide.
 8. The composition of claim 7, wherein the CMVpolypeptides that induce an antibody response consist of glycoprotein Band glycoprotein complex II, or antigenic fragments thereof.
 9. Acomposition comprising a plurality of sets of nucleic acid molecules,each set encoding a different type of human cytomegalovirus (HCMV)polypeptide that induces a cell-mediated immune response, and eachnucleic acid molecule of a set encoding the same type of HCMVpolypeptide.
 10. The composition of claim 9, wherein the polypeptidesthat induce a cell-mediated response comprises pp65 and pp150.
 11. Thecomposition of claim 7, wherein the polypeptides that induce aneutralizing antibody response are selected from the group consisting ofglycoprotein B, gM, gN, a combination of gM and gN (glycoprotein complexII; gcII), and a combination of gH, gL, and gO (glycoprotein complexIII; gcIII) of HCMV, and antigenic fragments thereof.
 12. Thecomposition of claim 9, wherein the polypeptides that induce acell-mediated immunity response comprise phosphoprotein 65,phosphoprotein 150, both phosphoprotein 65 and phosphoprotein 150, orantigenic fragments thereof.
 13. The composition of claim 7, wherein thepolypeptides that induce a neutralizing antibody response comprise gcIIor antigenic fragments thereof.
 14. The composition of claim 7, whereinthe polypeptides that induce a neutralizing antibody response comprisegcIII or antigenic fragments thereof.
 15. The composition of claim 7,wherein the polypeptides that induce a neutralizing antibody responsecomprise gB and gcII or antigenic fragments thereof.
 16. The compositionof claim 7, wherein the polypeptides that induce a neutralizing antibodyresponse consist of gB and gcIII or antigenic fragments thereof.
 17. Apharmaceutical composition that elicits an immune response against humancytomegalovirus (HCMV) comprising the composition of claim 1 and apharmaceutically acceptable carrier.
 18. A pharmaceutical compositionthat elicits an immune response against human cytomegalovirus (HCMV)comprising the composition of claim 7 and a pharmaceutically acceptablecarrier.
 19. A pharmaceutical composition that elicits an immuneresponse against human cytomegalovirus (HCMV) comprising the compositionof claim 9 and a pharmaceutically acceptable carrier.
 20. A method ofeliciting an immune response against human cytomegalovirus (HCMV) in asubject, the method comprising administering to the subject an amount ofa pharmaceutical composition of claim 17 effective to elicit an immuneresponse against HCMV in the subject.
 21. The method of claim 20,wherein administration is by needle injection, needle-less jetinjection, gene gun, topical administration, surgical administration, ormucosal administration.
 22. The method of claim 20, wherein the subjectis a non-human mammal or a human.
 23. The method of claim 22, whereinthe human is sero-negative for HCMV.
 24. The method of claim 23, whereinthe sero-negative human is selected from the group consisting of afemale between the ages of eleven and forty, a female contemplatingpregnancy, a pregnant female, an HIV-infected individual, a future organtransplant recipient, and a future bone marrow donor.
 25. The method ofclaim 22, wherein the human is sero-positive for HCMV.
 26. A kitcomprising the composition of claim 1 and instructions foradministration of the composition to a subject in an amount effective totreat a CMV infection.
 27. The kit of claim 26, wherein the amount iseffective to inhibit a future CMV infection.
 28. The kit of claim 26,wherein the amount is effective to treat an existing CMV infection. 29.The kit of claim 26, wherein the composition comprises DNA plasmids. 30.A kit comprising the composition of claim 7 and instructions foradministration of the composition to a subject in an amount effective totreat a CMV infection.
 31. A kit comprising the composition of claim 9and instructions for administration of the composition to a subject inan amount effective to treat a CMV infection.